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Ventilation in Amia calva : a comparison with water-breathing fish McKenzie, David J. 1990

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VENTILATION IN AM IA CALVA:  A COMPARISON WITH  WATER-BREATHING FISH. by DAVID J. MCKENZIE B.Sc., University of Dundee, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Zoology)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA June 1990 ©David J. McKenzie, 1990  In  presenting  degree freely  this  at the  thesis  in  partial  fulfilment  University  of  British  Columbia,  available for  copying  of  this  department publication  or of  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  Department of  Zoology  The University of British Vancouver, Canada  Date  DE-6 (2/88)  1  9  Columbia  t h June, 1990.  I further  purposes  the  requirements  I agree  gain shall  that  agree that  may  representatives.  financial  permission.  of  be  It not  the  by  understood be  allowed  an  advanced  Library shall  permission  granted  is  for  for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  i i  Aspects of ventilation and ventilatory control were investigated in an airbreathing fish, Amia calva, to determine the extent to which Amia is similar to water-breathing fish.  The possibility that Amia uses the air-breathing organ to maintain gas-exchange during periods of aestivation was tested.  During gradual air-exposure, Amia  showed no reduction in oxygen consumption, no increase in plasma urea levels or in urea excretion.  Arterial blood p H (pH ) remained constant, and arterial plasma a  total carbon dioxide (T  aCG2  ) and carbon dioxide partial pressure (P o2) increased. aC  Arterial plasma total ammonia ( T ^ ) and N H concentrations rose significantly. 3  Exposure to elevated total ammonia concentrations in the water did not elicit an increase in urea production or air-breathing.  Aquatic hypoxia without access to  air did not cause a reduction in aerobic metabolism and moderate levels were fatal.  These results indicate that Amia are incapable of aestivation, due to an  inability to reduce metabolism and detoxify ammonia to urea, and die following three to five days of air-exposure.  The air-breathing organ is used to maintain  aerobic metabolism under aquatic conditions of hypoxia or raised temperature. The characteristics of air-breathing and gill ventilatory responses to internal acid-base disturbances were investigated in Amia. Acid infusions lowered pH* and arterial blood oxygen content (C ), raised P a02  gill ventilation. raised P  aC02  aC02  , and stimulated air-breathing and  Ammonium bicarbonate infusions did not change p H or C  , and did not stimulate any ventilatory responses.  a  a 0 2  ,  Acid infusions  during aquatic hyperoxia lowered p H and raised P a  declined but remained above responses.  normoxic levels.  .  Arterial blood 0  There were  2  content  no ventilatory  These results indicate that air-breathing and gill ventilation responses  are most closely correlated with blood 0  in Amia  aC02  2  status, not p H or P 2. AiraC0  a  breathing and gill ventilation responses following acid infusion were associated with a release of stimulated  gill  catecholamines  ventilation  but  into  not  circulation.  Catecholamine infusion  air-breathing in  suggesting  Amia,  that  endogenous catecholamine release may have mediated gill ventilatory responses to hypoxaemia.  These ventilatory reflex responses to acid-base disturbance, and the  correlation between gill ventilation responses and catecholamine release are similar to observations made on water-breathing fish. Ventilatory responses to increases  in T  a C 0 2  and T ^ were investigated in  rainbow trout, and compared with responses by Amia. NaHC0  3  raised p H and T  ventilation.  a  a C 0 2  ,  did not change P  Infusion of N H H C 0 4  aCQ2  did not change p H  3  In trout, infusion of or C  and  stimulated ventilation.  stimulated ventilation.  a  a 0 2  , T  and stimulated  a Q 2  , raised T  a C D 2  , Pco2 a  Infusion of NH C1 lowered p H , raised T ^ , 4  a  Infusion of HC1 lowered p H , T a  a C 0 2  and C  a 0 2  , and  Infusion of NaOH raised p H but did not stimulate a  ventilation until twenty minutes post-infusion. effect on p H , C  ,  a  or C and T.^,,, and stimulated ventilation.  a Q 2  a C 0 2  or T  a m m  Infusion of NaCl had little or no  , and no effect on ventilation.  indicate that trout show a ventilatory response to increases in T T ^ and decreases in pF^ and C  a 0 2  a C Q 2  These results , increases in  , but not to increases in pH . Following HC1 a  and NaHC0 infusion, there was a significant increase in the level of circulating 3  iv catecholamines, indicating that the ventilatory responses to reductions in p H and a  C  a 0 2  The  and increases in T  a C 0 2  may be Immorally mediated by catecholamine release.  ventilatory responses to increases  catecholamine release.  in T ^ were not associated  Unlike trout, Amia  infusion of N H H C 0 , i.e. to increases in T 4  3  with a  do not show a ventilatory response to and T ^ . \  a C Q 2  Sites and afferent pathways for ventilatory reflex responses to blood and water  0  2  status were determined in Amia.  Air-breathing and gill ventilatory reflex  responses to hypoxia, sodium cyanide (NaCN), hypoxaemia and catecholamines were investigated  in intact Amia,  following  of branchial branches of cranial nerves  section  extirpation of the pseudobranch.  and compared with responses in animals IX and X , and  In intact, sham-operated animals, hypoxia  stimulated an increase in air-breathing and gill ventilation. Following denervation, the  air-breathing response  significantly attenuated.  was  abolished,  and the  gill  ventilation  response  In sham-operated animals, NaCN in the water flowing  over the gills stimulated air-breathing and gill ventilation, and NaCN given in the dorsal aorta stimulated gill ventilation. denervation.  These responses were abolished following  In intact animals, HC1 infusion stimulated air-breathing and gill  ventilation, but following denervation, the air-breathing response was abolished. The  ventilatory  response  to  catecholamines  denervated animals as compared with shams.  was  significantly  attenuated  in  These results indicate that air-  breathing and gill ventilation reflex responses are controlled by oxygen-sensitive receptors in the gills and pseudobranch, innervated by cranial nerves VII, IX and X.  These sites and afferent pathways are similar to receptors controlling hypoxic  V  reflex responses in water-breathing fish.  The effects of catecholamines on gill  ventilation are mainly exerted via stimulation of receptors in the gills, which are separate from those controlling air-breathing.  The gill ventilatory responses to  hypoxia, hypoxaemia and acidosis following denervation may be mediated by central effects of circulating catecholamines, or by an extrabranchial oxygen or p H receptor. In conclusion, Amia  is an entirely aquatic animal with the primary ventilatory  control mechanisms of water-breathing fish intact, but with the added ability to breathe air at the surface.  vi  ABSTRACT  T A B L E OF CONTENTS  ii  TABLE OF CONTENTS  vi  LIST OF TABLES  viii  LIST OF FIGURES  ix  LIST OF ABBREVIATIONS  xii  ACKNOWLEDGEMENTS  xiv  GENERAL INTRODUCTION  1  GENERAL MATERIALS AND METHODS  7  Chapter 1: Physiological responses to gradual air-exposure in Amia. INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION  15 16 18 25 54  Chapter 2: Ventilatory and Cardiovascular Responses to Blood pH, Plasma P 2> Blood 0 content and Catecholamines in Amia 59 INTRODUCTION 60 MATERIALS AND METHODS 62 RESULTS 66 DISCUSSION 84 C0  2  Chapter 3: Ventilatory and Cardiovascular Responses to Plasma Total C0 and Total Ammonia in Rainbow Trout and Amia 90 INTRODUCTION 91 MATERIALS AND METHODS 93 RESULTS 99 DISCUSSION 125 2  Chapter 4: The Effects of Branchial Denervation and Pseudobranch Ablation on Cardiovascular and Ventilatory Responses in Amia 131 INTRODUCTION 132 MATERIALS AND METHODS 134 RESULTS 139 DISCUSSION 165 GENERAL DISCUSSION  174  BIBLIOGRAPHY  viii  LIST OF TABLES Table 1: Best-fit linear regression equations and mean initial values of respiratory and blood gas variables under control aquatic conditions  26  Table 2: Best-fit linear regression equations and mean initial values of respiratory and blood gas variables during gradual air-exposure  32  Table 3: Best-fit linear regression equations for water variables during gradual airexposure.  40  Table 4: Best-fit linear regression equations and mean initial values of respiratory, blood gas and excretory variables during 900umol/l Table 5: Effects of HC1,  NH4CI  exposure  46  and HC1 in Hyperoxia on f , blood gases,  NH4HCO3  ab  [NE] and [E]  67  Table 6: Effects of NE and E on f and blood gases in normoxia and hypoxia, ab  and on [NE] and [E] in normoxia  79  Table 7: Blood Gas Measurements for Series 1 and 2  100  Table 8: Blood Gas Measurements for Series 3 and 4  110  Table 9: Plasma [NE] and [E]  Ill  Table 10: Normoxic Vo , pH , f , P and f  140  Table 11: Airbreathing frequency (breaths/hr)  141  Table 12: Arterial blood gases  142  2  a  h  op  g  IX  LIST O F F I G U R E S Figure 1: Individual plexiglass box with anterior air-space for air-breathing by Amia  10  Figure 2A: The relationship between V (t), V (a) and pH and Time (days) under 02  02  a  control aquatic conditions  28  Figure 2B: The relationship between T ^ , [NH ] and [urea] and Time (days) under 3  control aquatic conditions  30  Figure 3A: The relationship between V , pH and [urea] and Time (days) during Q2  a  gradual air-exposure  34  Figure 3B: The relationship between T  aC02  , [HC0 ] and 3  Pco2 and Time (days) a  during gradual air-exposure  36  Figure 3C: The relationship between T ^ and [NH ] and Time (days) during 3  gradual air-exposure  38  Figure 4A: The relationship between water pH and Pco2 and Time (days) during gradual air-exposure  42  Figure 4B: The relationship between water [NH ] and [urea] and Time (days) 3  during gradual air-exposure  44  Figure 5A: The relationship between V (t) and V 2(a) and Time (days) during Q2  900umolA NH C1 exposure  0  48  4  Figure 5B: The relationship between T (days) during 900umolA NH C1 exposure 4  amm  , [urea] and urea excretion and Time 50  Figure 6: Respiratory and blood gas variables at different levels of aquatic hypoxia  52  X  Figure 7: Representative traces of blood pressure and ventilation  69  Figure 8: Mean % change (± S.E.) in P , f , P and f following HC1 infusion. 71 DA  h  op  g  Figure 9: Mean per cent change (± S.E.) in P , f , P DA  h  and f following N H H C 0  op  g  4  infusion  3  73  Figure 10: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f following HC1 g  infusion during hyperoxia  76  Figure 11: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f following N E or E g  injection during normoxia  80  Figure 12: Mean per cent change (± S.E.) in P and f following NE or E infusion op  g  during moderate hypoxia  82  Figure 13: Mean per cent change (± S.E.) in Vg following NaCl, NaOH or HC1 infusion  103  Figure 14: Mean per cent change (± S.E.) in P  and f  op  following NaHC0 ,  g  3  NH^HCOs, HC1 and NaCl infusion.  106  Figure 15: Representative traces of ventilatory responses to NaHC0 , NH HC0 , 3  4  HC1 and NaCl in rainbow trout, and N H H C 0 and NaCl in Amia 4  3  108  3  Figure 16: Mean per cent change (± S.E.) in PDA» ftp Pop and f following NaCl g  infusion in rainbow trout.  113  Figure 17: Mean per cent change (± S.E.) in P , f , P DA  h  O P  and f following NaHC0 g  infusion in rainbow trout  3  115  Figure 18: Mean per cent change (± S.E.) in P , f , P DA  h  O P  and f  g  NH4HC0 infusion in rainbow trout  following 117  3  Figure 19: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f following HC1 g  XI  infusion in rainbow trout Figure 20: Mean per cent change (± S.E.) in P  119 D A  , f, P h  and f following NaCl  OP  g  infusion in Amia  122  Figure 21: Mean per cent change (± S.E.) in PDA) fh> Pop and f NHJHCOJ  following  g  infusion in Amia  124  Figure 22: The effects of aquatic hypoxia exposure on P  D A  , f, P h  and f in sham-  OP  g  operated and denervated Amia  144  Figure 23: Representative traces of cardiovascular and gill ventilatory responses to internal and external N a C N in shams and denervates  147  Figure 24: The effects of externally applied N a C N on P  D A  , f, P h  O P  and f in sham g  operated animals  149  Figure 25: The effects of externally applied N a C N on P  D A  , f, P h  and f  OP  denervated animals  151  Figure 26: The effects of N a C N given in the D A on P  D A  , f, P h  O P  and f in sham g  operated animals.  154  Figure 27: The effects of N a C N given in the D A on P  D A  , f, P h  and f  OP  in  g  denervated animals  156  Figure 28: The effects of N E . and E infusion on P  D A  , f, P h  and f  OP  g  in sham  operated animals Figure 29: The effects of N E and E infusion on P  in  g  159 D A  , f, P h  OP  and f in denervated g  animals Figure 30: The effects of acid and saline infusion on P denervated animals  161 D A  , f, P h  OP  and f  g  in 164  LIST OF ABBREVIATIONS  ABO:  Air-breathing organ  V :  Oxygen consumption  Vo (t):  Total oxygen consumption  V (a):  Oxygen consumption by air-breathing  ^C02  Carbon dioxide production  02  2  02  :  RE:  Respiratory exchange ratio  pH :  Arterial blood pH  a  T C02  :  a  Arterial plasma total carbon dioxide content  PaC02-  Arterial plasma carbon dioxide partial pressure  HCO/:  Arterial bicarbonate  Qo2  Arterial blood oxygen content  :  P a02  Pw02  T  Arterial plasma oxygen partial pressure  :  :  •  Water oxygen partial pressure Arterial plasma total ammonia content  NH :  Ammonia in the un-ionised form  DA:  Dorsal aorta  PDA  Dorsal aortic blood pressure  4:  Heart rate  3  :  P • •*• op  fr  Opercular pressure amplitude -  Gill ventilation rate Air-breathing frequency  Norepinephrine Epinephrine  xiv ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Dave Randall, for his support and guidance in my studies and while writing this thesis. I gratefully acknowledge the collaboration of Sumi Aota in Chapter 2, Hong Lin in Chapter 3 and Mark Burleson in Chapter 4. I  would like  further to  thank Mark  Burleson for his  patient  teaching  of  experimental techniques, and stimulating conversations. I thank all the members, past and present, of the Randall lab, for their help and pleasant company: Sumi Aota, Nick Bernier, Colin Brauner, Larry Fidler, Pat Gallaugher, George Iwama, Hong Lin, Dennis Mense, Bernice Miller, Mark Shrimpton, Yong Tang, Graeme Tolson, Bruce Tufts and Pat Wright. I was supported by a University Graduate Fellowship, by Dept. of Zoology Teaching assistantships, and by the N S E R C operating grant to D.J. Randall. I am especially continual support.  grateful to my parents, Iain and Anna McKenzie, for their  l GENERAL INTRODUCTION  All extant terrestrial vertebrates are considered to have evolved from freshwater piscine ancestors, which developed the ability to breathe air because it conferred on them selective advantages in hypoxic water.  The evolution of air-  breathing and a terrestrial lifestyle was not a single event, but all terrestrial vertebrates have evolved from the same group of aquatic vertebrates, as a result of similar selective forces (Randall, Burggren, Farrell and Haswell, 1981).  The  colonisation of land required more substantial changes in existing systems for gasexchange than simply the development of an ability to breathe air.  The three  main respiratory gases in fish are oxygen, carbon dioxide and ammonia. There are differences between air and water as respiratory media, as listed below, and the successful colonisation of land required different adaptations to control levels of respiratory gases in the body fluids.  Studying respiratory adaptations in extant  air-breathing fish may well give insight into the changes in respiratory control systems associated with the colonisation of land. Water has a low capacitance for oxygen, and so in order to meet the oxygen requirements of metabolism, water-breathing fish ventilate large volumes of water across their gills.  The capacitance of water for carbon dioxide is high, and so all  carbon dioxide produced by metabolism is effectively flushed out of the animal across the gills, resulting in carbon dioxide tensions in the blood that are typically very low (Dejours, 1981). The result of these differential capacitances is that the primary source of respiratory drive in water-breathing fish is oxygen, and there  2  appears to be little sensitivity to carbon dioxide (Smith and Jones, 1982, Shelton, Jones and Milsom, 1986). The capacitance of water for ammonia is even higher than that for carbon dioxide, and so ninety percent of ammonia produced by protein catabolism is also effectively flushed out of the animal across the gills, and about ten percent voided in the urine (Randall and Wright, 1987; Randall, 1990). The result of this is that freshwater fish excrete the majority of their nitrogenous waste as ammonia (Randall and Wright, 1987; Randall, 1990). The oxygen content of air is high, so animals with the capacity to breathe air do not have to ventilate to the same extent as water-breathers to meet metabolic oxygen requirements.  The oxygen and carbon dioxide capacitances of air are  equal, so changes in ventilation will change the amount of carbon dioxide in the body fluids, and therefore acid-base status (Rahn, 1966; Rahn and Howell, 1976; Dejours, 1981). Thus there was undoubtedly selection pressure in favour of airbreathers that were able to monitor both body fluid oxygen and carbon dioxide or pH levels, and adjust ventilation to maintain homeostasis.  This is an ability that  all extant vertebrate air-breathing groups possess (as reviewed by Dempsey and Forster, 1982; O'Regan and Majcherczyk, 1982; Scheid and Piiper, 1986; Shelton, Jones and Milsom, 1986; Smatresk, 1990). The capacitance of air for ammonia is extremely low, and so ammonia, which is toxic in all vertebrates, would tend to accumulate very quickly in air-breathers. The requirements of water balance on land are such that continual water loss to remove ammonia would not be sustainable.  This would exert intense selection  3  pressure in favour of those animals that were able to detoxify ammonia, and accumulate non-toxic wastes.  Thus, all terrestrial vertebrates have the ability to  detoxify all nitrogenous waste as urea or uric acid (Smith, 1961), an adaptation that is absent in most extant water-breathing, bony fish (Mommsen and Walsh, 1989) . While amphibians are known to show ventilatory sensitivity to carbon dioxide and p H (Maclntyre and Toews, 1973; Ishii, Ishii and Kusakabe, 1985; Smatresk, 1990) , there is very limited information about ventilatory control in air-breathing fish, and the extent to which such control systems are similar to air-breathing or water-breathing systems.  It is likely that air-breathing fish are more similar to  water-breathers, because the air-breathing organ (ABO) in these animals is used to supplement oxygen uptake, and the gills are used in carbon dioxide excretion (Johansen, 1970).  It is probable that ventilatory sensitivity to carbon dioxide  developed when the lung became a site for carbon dioxide excretion into air, as is the case in amphibians (Randall, 1974; Maclntyre and Toews, 1973). A  capacity to detoxify  ammonia as urea by the ornithine cycle used by  terrestrial vertebrates is found in all elasmobranchs, but only in a number of bony fish, for example as an adaptation to survive air-exposure by a air-breathing fish (Saha and Ratha, 1986) or alkaline waters in a water breathing fish (Randall, Wood, Perry, Bergman, Maloiy, Mommsen and Wright, 1989).  As stated earlier, oxygen is the primary ventilatory stimulus in water-breathing fish (Smith and Jones, 1982; Randall, 1982; Shelton et al,  1986).  There is  4 evidence, however, that fish show a ventilatory response to increases in plasma carbon dioxide content, when associated with a plasma alkalosis (Janssen and Randall,  1975), and mammals are known to show a ventilatory response  ammonia (Wischer and Kazemi, 1974).  to  Ventilatory responses to increases in  plasma carbon dioxide and ammonia content have not been investigated in either water-breathing or air-breathing fish.  Increases in ventilation might function to  remove excesses of endogenously produced carbon dioxide or ammonia.  Despite considerable differences in respiratory physiology, the neuro-hormones norepinephrine and epinephrine exert effects on ventilation in all vertebrate groups.  Infusion  of catecholamines  stimulates  ventilation in several  water-  breathing fish (Peyraud-Waitzenegger, 1979; Aota, pers. comm.), and in mammals (Dempsey, Olson and Skatrud, 1986).  Furthermore, there is evidence to suggest  that catecholamines are involved in respiratory control in both water-breathing fish and in mammals, via effects on peripheral chemoreceptors and the central nervous system (Dempsey  et ah,  1986;  Randall and Taylor,  1989;  Aota, Holmgren,  Gallaugher and Randall, 1990). There is no information about the possible role of catecholamines in gill ventilation and air-breathing responses in air-breathing fish. Ventilatory responses to increases  in blood pH, carbon dioxide  content  or  ammonia might be mediated by circulating catecholamines.  In mammals, peripheral receptors affecting ventilation are in the carotid and aortic bodies (O'Regan and Majcherczyk, 1982).  In reptiles and amphibians,  5 oxygen and earbon dioxide sensitive receptors have also been identified in the blood vessels leaving the heart (Ishii, Ishii and Kusakabe, 1985a,b).  These areas,  for amphibians, reptiles and mammals, are anatomically homologous with the gill arches of fish (Romer, 1970).  Indeed oxygen sensitive receptors have been  identified in the first gill arch of fish (Milsom and Brill, 1986; Burleson and Milsom, 1990).  The site of peripheral receptors controlling gill ventilation and  air-breathing responses in air-breathing fish are unknown, although there  is  evidence that they are in the gills (Smatresk, 1986; Smatresk, 1987).  Amia  calva  is an Actinopterygian fish, the only extant species in the sub-  division Halecomorpha. It is a primitive fish, only distantly related to teleosts and other extant bony fishes; with fossil remains dating from the Jurassic (Nelson, 1984).  Amia  is piscivorous, and an active predator.  It occurs in shallow, slow-  moving freshwater in eastern North America, from southern Ontario and Quebec to Texas (Scott and Crossman, 1973).  Amia  provides a readily available and  interesting animal model of an intermediate stage in the evolution of air-breathing and survival on land.  It possesses both gills  for water-breathing  swimbladder adapted to function as an air-breathing organ.  and a  The gills have an  unusual structure, to prevent collapse in air (Daxboeck, Barnard and Randall, 1981; Olson, 1981), and there are anecdotal reports (Dence, 1933; Neill,  1950)  that it is capable of surviving prolonged periods of air-exposure by aestivating. This apparent ability to aestivate, and the implication that Amia  can detoxify  ammonia as urea in a manner similar to the African lungfish (Smith, 1930; Smith,  6 1961), has never been tested.  It is also unknown whether the respiratory  physiology of Amia is similar to purely water-breathing fish, or whether it displays some characteristics of air-breathing vertebrates. This thesis examines the responses of Amia  calva  to gradual air-exposure, to  determine whether they aestivate in a manner similar to the lungfish (Smith, 1930; Delaney, Lahiri and Fishman, 1977).  The possibility that Amia  shows ventilatory  responses to changes in blood p H and carbon dioxide tension is tested, and the ventilatory  responses  to  catecholamines  assessed.  Ventilatory responses  to  increases in plasma carbon dioxide and ammonia content are investigated in waterbreathing fish (trout) and compared with responses seen in Amia,  and a possible  role for circulating catecholamines in the observed responses is discussed. site of peripheral chemoreceptors  The  controlling gill ventilation and air-breathing  responses, and the site of catecholamine stimulation of ventilation is determined in Amia.  The results of these experiments are discussed to assess the extent to  which Amia  is similar to water-breathing or air-breathing vertebrate groups.  7 GENERAL MATERIALS AND METHODS.  Experimental animals: Amia  calva  were netted in Lake Erie, southern Ontario, Canada. Animals were  air-freighted to U.B.C., in large, heavy duty bags containing one-third water and two-thirds 100% 0 . 2  Amia  Mortality from transit was less than one percent.  At U.B.C.,  were maintained in large outdoor circular fibreglass tanks, with a constant  flow of dechlorinated Vancouver tapwater (Temperature = 7 to 12°C, p H approx. 6.5). libitum,  The animals were fed live goldfish, trout fry or salmon fry, usually adand at least once per week.  A l l animals were allowed a minimum of  three weeks recovery following transit, before experimentation. A l l experiments were conducted at 20°C. Amia were placed in small plexiglass holding tanks, and the temperature raised to 20°C over a minimum of three days (usually five). Rainbow trout were obtained from West Creek Trout Farms, Aldergrove, B . C . and maintained in large, outdoor circular fibreglass tanks with a constant flow of dechlorinated Vancouver tapwater, at a temperature of 7 to 12°C. Animals were fed trout chow daily, and starved for at least 48h prior to use in any experiments.  Cannulations: Fish were fitted with chronic indwelling cannulae in the dorsal aorta and, when required, in the operculum, under general anaesthesia in short operations lasting 15 to 20 minutes.  A l l fish were anaesthetised in Tricaine Methane Sulphonate  8 (MS222) at concentrations of 1:10,000 until ventilatory movements ceased, and then placed ventral side up on an operating table where the gills were irrigated with an MS222 solution at 1:20,000. Dorsal aortic cannulations were performed using the technique of Soivio, Westman and Nyholm (1972).  A sharpened wire was inserted into PE-50 tubing  such that only the tip protruded at the end.  A blind puncture was made in a  caudal direction (at a 45 degree angle) in the midline of the branchial basket, between the first and second gill arches.  The wire punctured the wall of the  dorsal aorta, and then was used to guide the tubing into the vessel. The wire was removed, and the tubing advanced for three to five centimetres.  The cannula was  secured to the roof of the mouth with a suture, and led out of the roof of the mouth in front of the nares, via a flanged section of tubing (PE-200). Opercular cannulation was performed by drilling a hole in the centre of the operculum, and feeding a flanged section of tubing (PE-200) through from the inside.  A flanged cuff was then attached on the outside, to hold the cannula  tightly in place. All fish were allowed to recover from surgery for 48h in individual perspex boxes, with an ample flow of water. Amia  were allowed to recover in boxes with  an anterior air-space (fig 1), to allow air-breathing.  Dorsal aortic cannulae were  flushed daily with heparinised (10,000 USP units/L sodium heparin) Cortland's saline (Wolf, 1963).  9  Figure 1: Individual plexiglass box with anterior air-spaee for air-breathing by  Amia.  10  11  Measurement of blood and plasma variables: Arterial blood pH was measured using a Radiometer microelectrode (E5021) and acid-base analyser (PHM 72) thermostatted to the temperature of the fish, and calibrated with Radiometer precision phosphate buffers S1500 and S1510. Arterial blood P  0 2  was measured on anaerobically collected blood samples  using a Radiometer electrode (E5G46) and acid-base analyser, thermostatted as for pH . a  The 0  2  electrode was calibrated with water-saturated N and air. 2  Arterial blood 0  2  content was measured using a Radiometer electrode (E5046)  calibrated with 0 -free sodium sulphite, and air-saturated water, and the method 2  described by Tucker (1967), at the fish temperature, using 30 (il blood samples collected anaerobically in a gastight Hamilton syringe. Plasma for T  a C 0 2  determination was obtained by centrifuging (Damon/TEC)  blood, immediately upon collection, in heparinised microhaematocrit capillary tubes, and withdrawing 25 |il of plasma into a gastight syringe.  25 jxl of plasma  was shaken for three minutes with 1 ml of 0.1 M HC1 and 7 ml of 100% N in a 2  10 ml gastight syringe, to liberate all the G 0 equilibration of the gas and liquid phase.  2  into the gas phase and ensure  At least 5.5 ml of the gas was injected  via a drying filter into a 1 ml sample loop of a gas chromatograph (Carle GC100) with a Poropak Q C 0  2  calibrated with 10 m M T  discriminating column. C 0 2  standards. The T  a C Q 2  The gas chromatograph was was calculated by integrating the  signal from the gas chromatograph with a data acquisition card (Data Translation 2801) and an Olivetti M24 computer.  12  Plasma for  and urea concentration ([urea]) determination was obtained by  centrifuging whole blood samples in 1.5 ml micro test-tubes (Eppendorf) with a micro-centrifuge (Fisher, Model 235), within five minutes of collection. was stored on ice for a maximum of 30 minutes.  Total ammonia concentration  was determined colorimetrically with a U V spectrophotometer 160), and a Sigma kit.  Plasma  (Shimadzu U V  Plasma [urea] was determined by incubating plasma with  urease (Boeringher) and then analysing the samples with the Sigma ammonia kit. Plasma samples for catecholamine  analysis were collected by centrifuging  whole blood in a micro-centrifuge (Fisher, Model 235), decanting plasma, and immediately freezing in liquid N . 2  Catecholamine concentrations ([NE] and [E])  were determined on alumina-extracted plasma samples by high performance liquid chromatography (HPLC) with electrochemical detection, using a Waters Plasma Catecholamines reverse-phase column, Waters M460 Electrochemical Detector and Waters 510 solvent delivery pump (Waters/Millipore), as described by Woodward (1982)  and Primmett, Randall,  Mazeaud and Boutilier  generated on a chart recorder (Soltec 1241).  (1986),  with  peaks  Catecholamine concentrations were  calculated by integrating the area under peaks with Sigmascan (Jandel Scientific) and an Olivetti M24 computer, and comparing with peaks from D H B A , N E and E standards.  13  Measurement of water and air variables: Water p H was measured using a Radiometer combination p H electrode (GK 2402B) and a Radiometer acid-base analyser (PHM 72).  The electrode  was  calibrated with standard Radiometer buffer solutions at p H 7 and 4. Water and air P  0 2  were measured as for P  a02  i but with large (5 to 10 ml)  samples in gastight syringes. Water T  C 0 2  was measured as for T  a C 0 2  , but 1 ml of water was mixed with 1 ml  0.1M HC1 and 9 ml 100% N in a gastight syringe.  The gas-chromatograph was  2  calibrated with 0.5mM T  C Q 2  standards.  Water total ammonia concentration was measured colorimetrically with a U V spectrophotometer  (Shimadzu  UV  160)  using  a  micro-modification (D.G  MacDonald, pers. comm.) of the technique of Verdouw, van Echteld and Dekkers (1978). Water [urea] was measured using a micromodification ( C M . Wood, pers. comm.) of the Crocker (1973) technique.  Calculations: Plasma P 2 was calculated using the Henderson-Hasselbalch equation: C0  (aCo2) . (1 + antilog (pH - pK))  Apparent pK and ocC0 values for trout plasma were used as calculated for the 2  correct temperature in Boutilier, Heming and Iwama (1984), for trout, and for  14 Amia the apparent pK and a C 0  2  for gar (Lepisosteus osseus) plasma as calculated  at 20°C in Smatresk and Cameron (1982) was used. Plasma  NH  3  ([NH ]) 3  concentration  was  calculated  with  the  Henderson-  Hasselbalch equation: Plasma [NH ] = 3  T  a m m  . (antilog pH„ - pK)  1 + (antilog p H - pK) a  Plasma pK was calculated from values given for Oncorhynchus  mykiss in  Cameron and Heisler (1983). Water P  was calculated using the Henderson-Hasselbalch equation, and the  C 0 2  apparent p K and a C 0 as calculated (for the experimental temperature) in Boutilier 2  et al (1984). Water [NH ] was calculated as for plasma [NH ], using the pK at the correct 3  water temperature from Boutilier et al (1984).  3  Chapter 1 : Physiological responses to gradual air-exposure in  Amia.  16 INTRODUCTION  There are reports in the literature that Amia can survive prolonged emersion by aestivating, in a manner similar to the African lungfish, Protopterus sp. (Smith 1961).  Dence (1933) found an Amia living in a mud puddle, in northeastern  U.S.A. (New York) and the animal quickly burrowed into the substrate when disturbed.  In southeast U.S.A. (Georgia), Neill (1950) found an Amia in a  spherical underground chamber, at some distance from a recently flooded river. The animal was apparently in good health. To aestivate, an animal must be able to reduce water loss, avoid a toxic accumulation of wastes and rely on air-breathing for gas exchange.  Avoiding  desiccation requires that ventilation, and therefore oxygen uptake, be reduced, leading to a reduction in aerobic metabolism. energy stores, since feeding is impossible. water losses.  This also allows conservation of  Burrowing further reduces evaporative  Detoxification of ammonia to urea allows the animal to store  nitrogenous wastes, avoid ammonia toxicity and reduce urine volumes.  Thus, if  Amia aestivate, they must be capable of reductions in aerobic metabolism and of converting ammonia to urea. In the present study, Amia were gradually air exposed over a ten day period, and various respiratory and internal variables measured to determine whether they aestivate. aestivation,  It is possible that emersion is not an adequate stimulus to initiate so  two  further experiments  were  performed.  Elevated aquatic  ammonia levels are known to stimulate increases in urea production in the  17 goldfish (Carassius auratus) (Olson and Fromm 1971), and an increase in water borne irritants leads to an increase in air-breathing in gar (Lepisosteus osseus) (Smatresk 1988). If an Amia were trapped in a gradually evaporating mud puddle, a build up of ammonia in the water, rather than dehydration, might stimulate an increase in urea production and air-breathing.  Thus, Amia were exposed  to  elevated aquatic ammonia levels, and air and water breathing and urea production monitored.  In sturgeon (Acipenser  transmontanus) aquatic hypoxia elicits a  reduction in aerobic metabolism (Burggren and Randall 1979) and the same is true in trout (Oncorhynchus mykiss) (Boutilier et al. 1988).  Amia were exposed to  differing degrees of aquatic hypoxia, without access to air breathing to supplement their oxygen metabolism.  uptake, to determine if this resulted in a decrease in aerobic  18 MATERIALS AND METHODS.  Experimental animals: Amia  calva  weighing  between  300  and  1000  g  were  maintained and  temperature acclimated as described in General materials and methods.  Animal preparation: Following three to five days at 20°C, the animals were anaesthetized in a buffered (NaHC0 ) tricainemethanesulphonate (MS222) solution at a concentration 3  of 1:10,000 and transferred to an operating table, where they were maintained at a MS222 concentration of 1:20,000.  Dorsal aortic cannulae (PE50) were implanted  using the technique of Soivio et al. (1972). The fish were then left to recover for 48 hours in the plexiglass holding tanks.  Experimental protocols: 1 ) Control Measurements: Animals were placed in individual black plexiglass boxes (volume = 9 1) with access to a forward air space for air-breathing (volume 1.4 1), and allowed 24 hours to recover.  Following recovery, a 1 ml blood sample was removed,  replaced with an equal volume of heparinised (1:1,000) Cortland's saline (Wolf 1963), and pH , T a  a m m  and plasma urea concentration ([urea]) measured as described  in General materials and methods. decline in P  o z  The forward airspace was then sealed, and the  as a result of air-breathing by the Amia measured over a two hour  19 period, following which the space was re-opened. Samples of inflow and outflow water were also analysed for P two to three day intervals.  w 0 2  .  The blood sampling regime was repeated at  This protocol was followed for a minimum of ten  days.  2) A i r Exposure: Following  the post-surgical recovery period, while  still in the  plexiglass  holding tank, 1.5 ml blood samples were collected anaerobically, in a gastight syringe (Hamilton), and replaced with an equal volume of heparinized saline. Arterial blood pH, T  a C 0 2  , T ^ and plasma [urea] were measured, as described in  general materials and methods. The animals were then placed in black plexiglass chambers (volume approx. 120 1) containing a known volume of water (approx. 50 1) and a substrate of either washed river sand or 1/8" mesh plastic netting slung between bags of washed river sand.  A control chamber containing the same substrate and water  volumes, but no fish, was also prepared.  The chambers were placed at a slight  diagonal inclination using ramps of bagged sand, and the water circulated via an inlet at the topmost corner of the chamber lid and an outlet at the bottommost corner of the chamber.  The water was circulated from experimental to control  chamber, and vice-versa, using a peristaltic pump (Watson Marlowe M R H E 100 using Marprene 0.5  cm I.D. tubing).  approximately 12 litres per hour. produce an airtight seal.  Water flow  rate was maintained at  The lids of the chambers could be closed to  The water in control and experimental chambers could  20 be circulated separately and mixed samples of both water and air in the chambers removed to monitor changes in P . 02  Water volume was decreased at a rate of approximately four litres per day, from both control and experimental chambers, so that the fish were completely air exposed at ten days (exact volumes of water removed differed slightly for each fish, as a result of differing initial volumes). day, and pH,  and [urea] measured.  Water samples were taken every  Every third day, a 1.5 ml blood sample  was collected anaerobically, and replaced with an equal volume of heparinized saline.  Arterial blood p H , T  a C 0 2  , T ^ , and plasma [urea] were analysed.  If the  fish's cannula was not patent, then an attempt to remove a blood sample was made the following day.  Every attempt was made to disturb the animal as little  as possible during sample collection. fish  in the  experimental  Twenty four hours after first placing the  chamber, or blood sampling, water chambers was  separated  and the  flow  between control and  chambers  sealed.  Water (a  minimum of 3 x 5 ml) and air (a minimum of 3 x 10 ml) samples were removed from each chamber and analysed for P  0 2  and T  C Q 2  .  Based on measurements of V  D 2  under control conditions, the experimental chamber remained sealed long enough to produce an approximately 20 mmHg decline in P , and then additional water 02  and air samples were removed, P  0 2  measured, and the chamber unsealed.  Once the animal was completely air-exposed, water removal was stopped but blood, air and water sampling continued, as described, until the fish's death. 3) Ammonium Chloride exposure: Fish were placed in individual black plexiglass boxes identical to those used  21 for control measurements.  24 hours later, a 1 ml blood sample was removed, and  replaced with an equal volume of heparinized saline. plasma [urea] were measured.  Arterial blood pH,  and  Daily measurements were made of inflow and  outflow water P , and of the decline in P 02  G 2  in the forward chamber over a two  hour closure period. The flow of water through the boxes was then shut off, and water samples removed and analysed for [urea].  Two hours later, further samples  were collected and the same parameter measured. Flow was then resumed. Following the removal of control blood, air, and water samples, ammonium chloride (NH C1) was pumped into a large header tank at a constant rate, where it 4  was mixed with incoming water, so that the fish were exposed to water with an NHjCl concentration of 923 ± 9 (mean ± S.E.) umol/1.  Water pH was 6.68  water [NH +] was 921 umol/1 and water [NH ] was 1.9 umol/1. 4  3  NHtCl concentrations led to over 50% mortality.  ±0.1,  Slightly higher  The water and air sampling  protocol described above was followed daily for 10 days of exposure to NH C1. 4  Every second day, the blood sampling regime was repeated. Eight fish were put through the above protocol, but all animals no longer had patent cannulae after four days.  In order to obtain blood readings from animals  later in the exposure regime, fish were exposed to the NH C1 for three to eight 4  days, and then chronically cannulated.  Cannulation was by the same method as  desribed earlier, but fish were anaesthetized in water with MS-222 and 923 ± 9 mmol/1 NH4CI, and irrigated during surgery with water at the same NH4CI concentration.  Following a 48  withdrawn and analysed for T  a m m  hour recovery period, blood samples  and [urea].  were  22  4 ) Hypoxic Exposure: Amia were placed in individual black plexiglass boxes (volume = 61), without access to an airspace, and with a water flow rate of approximately 500 ml/min. 24 hours later, samples of inflow and outflow water were analysed for P T  C 0 2  0 2  and  , and a 1 ml blood sample withdrawn anaerobically, replaced with an equal  volume of heparinized saline, and pH , T a  The P  Q2  a C 0 2  , and  measured immediately.  of the inflow water was then reduced using a gas exchange column  with nitrogen gas flowing counter-current to water flow. reduced to one of four levels: 111+0.59 mmHg, 85.2±2.37 mmHg, or 30±0.22 mmHg.  The water P  0 2  was  mmHg, 59.3+0.34  Following 24 hours' exposure to one of these P  levels, samples of inflow and outflow water were analysed for P  0 2  and T  c 0 2  w02  , and a  1 ml blood sample withdrawn, replaced with saline, and the relevant variables measured.  Analytical methods:. During air exposure, V  0 2  was calculated, given the change in P  0 2  in the boxes  while sealed, the time elapsed, the water and air volumes, and the weight of the fish.  Oxygen consumption was expressed as mg/kg/hr, and corrected for any  background oxygen  consumption as measured in the control box.. - Oxygen  V  were calculated in aquatic hypoxia, using the Fick principal  consumption and  c02  and the values of P  0 2  and T  C 0 2  for inflowing and outflowing water.  Oxygen  consumption under control conditions and during ammonium chloride exposure  23 was calculated in the same way for the water phase (V (w)). 02  Under all these  circumstances, inflow and outflow gas tensions were in steady state.  V  0 2  in air  (V (a)) was calculated knowing the volume of the air space, the decline in P 02  during closure, the closure time and the fish's weight.  Total V  Q 2  0 2  (V (t)) was 02  calculated by adding water and air quantities. Water and plasma P  were calculated using the Henderson-Hasselbalch  C 0 2  equation, as described in general materials and methods.  Plasma bicarbonate  concentration ([HC0 "]) was calculated using the following equation: 3  plasma [ H C 0 ] = T 3  a C Q 2  - (a C 0 . P 2  aC02  )  Plasma and water [NH ] were calculated using the Henderson-Hasselbalch 3  equation, as described in General materials and methods. Urea excretion rates during ammonium chloride exposure were calculated given the initial and final water urea concentrations, the closure time, the box volume and the fishes' weight.  24  Statistical analysis: All  measured variables under control, air exposure  and NH4CI  exposure  conditions were plotted against time, and the relationship described with a best-fit linear regression.  The regression coefficient of each variable was compared with  a coefficient of zero, and the regression coefficients of the control variables were compared with the regression coefficients  of the same variables during air or  NH4CI exposure, using a modification of the Student's T-test (Zar 1984).  The  least squares fit of the regression to the data was not improved by using second or third order regressions.  The regression coefficients of T  a C 0 2  , P  and [ H C 0 ]  a C 0 2  3  during air exposure were compared in the same way. The mean + S.E. was calculated for the initial ("day 0") values of the control variables, and compared with the same variables during air and NH C1 exposure 4  using T-tests. P<0.05 was taken as the fiducial limit of significance. For the hypoxic exposure experiment, measured variables at each level of P  w Q 2  were compared using the Kruskal-Wallis test for non-parametric analysis of variance.  In those cases where there was a significant (p < 0.05)  amongst variables at different P ^  levels,  difference  a modification of the Tukey "a  posteriori" test (Zar 1984) was used to compare the control and experimental conditions.  25  RESULTS  Gradual air exposure on a substrate of autoclaved alluvial mud (n = 4), sterile potting soil (n = 3) or washed river sand (n = 6) did not result in any attempts to burrow by the Amia.  During gradual air-exposure the fish created a shallow  depression in the substrate by moving their body from side to side.  Neither air  exposure nor NH C1 exposure led to an increase in urea production or excretion. 4  Air exposure and aquatic hypoxia without access to air did not elicit any reduction in V  G 2  .  1) Control: The best fit linear regression equations, R and mean initial values ± S.E. of 2  the control variables are in Table 1. on all variables measured. variables  showed  a  Figures 2A and B show the effects of time  None of the regression coefficients  significant  difference  from  zero,  of the control  indicating  that  these  parameters remained constant over time.  2 ) Air-exposure: A l l the fish survived at least 24 hours of complete air exposure, and most animals survived for three to five days. Following complete air-exposure, the  26 Table 1 : Best-fit linear regression equations and mean initial values of respiratory and blood gas variables under control aquatic conditions.  R.C.S.E.  meaittSE  G.01  0.72  61.5±13.4  y=0.31x+7.27  0.05  0.20  3.9±2.8  31  y=0.08x+6.91  0.06  0.06  7.70±0.03  6  28  y=1.40x+312.6  .001  0.04  314.6±55.7  [NH ]  6  25  y=-0.15x4-5.9  0.09  -0.3  6.1±1.4  [urea]  6  27  y=.51x+121.7  0.08  -0.28  108.6±2.9  Variable  N  n  Best-fit  R  V (t)  6  45  y=.51x+63.04  V (a)  6  49  pH  6  Q2  G2  a  T  •*• amm  3  2  N = number of fish, n = number of observations. R.C.S.E. = regression coefficient standard error. Units: V (t) and V (a) = mg/kg/hr; T 02  02  a m m  , [NH] and [urea] = umol/1.  27  Figure 2A: The relationship between V (t), V (a) 02  02  and p H and Time (days) under control a  aquatic conditions, n = 6 Each symbol represents an individual animal.  200  150  100  d • W  o  -A  50  (mg/  'kg/ 'hr)  -+40-  20-  • A  o  A  i  o •>  a  •  •  • •  D  Q  •  1  O  •  O  •  8.500--  8.000 - -  II ^  7.500--  A. O  r  •4-B-s A  •  7.000 10  Time (days)  12  14  16  29  Figure 2B: The relationship between T ^ , [NH ] 3  and [urea] and Time (days) under control aquatic conditions, n = 6 Each symbol represents an individual animal.  Time (days)  31 animals continued to make gill ventilation movements, breathing behaviour.  interspersed with air-  At death, most animals had a characteristically bloated  appearance, suggesting over-inflation of the swimbladder. The relationship between time (days), and measured respiratory and blood gas variables during gradual emersion can be seen in figures 3A, B and C . The best fit linear regression equations, R and mean initial values ± S.E. of these variables 2  are shown in Table 2.  The mean initial values for V  0 2  , p H , T ^ , and plasma  [NH ] were not significantly different from control values.  The mean initial value  3  for plasma [urea] was  significantly  a  higher in the animals that  underwent air exposure than in the control animals. Vo2 during gradual air-exposure, indeed, V  Q 2  subsequently  There was no reduction in  rose slightly as the Amia gradually  became emersed, and the regression coefficient was significantly different from zero (fig 3A).  However, the regression coefficient during gradual emersion was  not significantly different from the regression coefficient  derived for control  measurements. Arterial blood p H and plasma [urea] (fig 3A) did not change during gradual emersion.  Arterial plasma total C 0 , P 2  and [HC0 ~] (fig  a C 0 2  3  3B) all rose  significantly during air exposure, especially following complete air-exposure at 10 days.  The regression  coefficients  for  T  a C 0 2  ,  P  a C 0 2  and  [HCOy]  were  significantly different from each other, all increased to the same extent.  not  Arterial  plasma total [ammonia] and plasma [NH ] both increased significantly during 3  gradual emersion (fig 3C), especially following complete air-exposure at 10 days.  32  Table 2: Best-fit linear regression equations and mean initial values of respiratory and blood gas variables during gradual air-exposure.  R.C.S.E.  mean+SE  Variable  N  n  Best-fit  R  v  7  30  y=2.6x+63.61  0.13  1.25  77.8±9.4  8  34  y=0.01x+7.71  0.13  3.34  7.72±0.01  T»C02  6  24  y=0.76x+12.13*  0.48  0.17  11.8±0.57  PaC02  6  24  y=0.79x+8.34*  0.32  0.25  7.76+0.39  [HCOy]  6  24  y=0.72x+11.7*  0.48  0.16  11.40±0.55  T  9  38  y=60.x+251.0*  0.24  13.9  383.1±59.0  [NH ]  9  36  y=0.67x+4.9*  0.31  0.17  6.7+1.0  [urea]  6  25  y=1.80x+315.8  0.01  4.08  304.3±53  0 2  pH  a  imm  3  2  N = number of fish, n = number of observations. R.C.S.E. = regression coefficient standard error Units: V  Q 2  = mg/kg/hr; T  a C 0 2  and [HCOy] = mmolA; P co = mmHg; T ^ , [NH ] a  2  and [urea] = umol/1. * = significantly different from zero and/or control regression.  3  Figure 3A: The relationship between V  o z  , pH  and [urea] and Time (days) during gradual air-exposure Each symbol represents an individual animal.  a  Time (days)  Figure 3B: The relationship between T and P  a C 0 2  a C 0 2  , [HCOy],  and Time (days) during gradual air-exposure  Each symbol represents an individual animal.  Time (days)  Figure 3C: The relationship between T  a m m  and [NH ] and Time (days) during gradual 3  air-exposure. Each symbol represents an individual animal.  38  2500  o  E E E o  i—  + n=9  2000--  1500--  rr  1000--  D  E V) a CL  o E  500  i  0  O  o  O-  V  ! 9  1  - n=9 •  E  CO _D QL  •  • •  D  o  :  O  .  *•  ;  r1  I " ° •I 1  —  1  V  L  '  1  10  12  14  t  air—exposue  Time (days)  16  The relationship between time (days) and water pH, P  C02  , [NH ], and [urea] can 3  be seen in figures 4A and B. The best fit linear regression equations and R values are in table 3.  Water P C0  2  C 0 2  2  Water p H did not change significantly during air exposure.  rose initially, and then reached a new equilibrium between excretion of  by the fish and diffusive loss to the atmosphere.  for water P  c o 2  The regression coefficient  was not significantly different from zero, indicating that the C 0  2  diffusion gradient between plasma and water increased significantly as the fish became emersed. vice-versa.  Water T  C 0 2  was dependent on water pH, rising as p H rose and  Water [NH ] rose greatly during three experiments but remained 3  constant during two, the combined data leading to a regression coefficient that indicated a significant increase.  Increases in plasma [NH ] were correlated with 3  increases in water [NH ] up to air exposure, in those individuals in which both 3  parameters were measured simultaneously. Following air exposure, however, there was no clear relationship between water and plasma [NH ] levels. 3  Water [urea]  increased significantly during air exposure, but translation of daily water [urea] measurements into daily excretion rates yielded variable results that did not indicate a significant increase in urea excretion.  40  Table 3: Best-fit linear regression equations for water variables during gradual air-exposure. Variable  N  n  Best-fit  R  pH  7  78  y=0.04x+6.44  0.02  0.02  Pc02  7  32  y=0.03x+7.32  0.003  0.05  [NH ]  5  60  y=0.61x-1.74*  0.39  0.10  [urea]  4  48  y=3.58x+13.6*  0.36  0.70  3  N = number of fish, n = number of observations. R.C.S.E. = regression coefficient standard error. Units: P  C 0 2  = mmHg, [NH ], [urea] = u.mol/1. 3  * = significantly different from zero.  2  R.C.S.E  41  Figure 4A: The relationship between water p H and P 2 and Time (days) during gradual C0  air-exposure. Each symbol represents an individual animal.  Time (days)  43  Figure 4B: The relationship between water [NH ] 3  and [urea] and Time (days) during gradual air-exposure. Each symbol represents an individual animal.  30  n =  5  20--  10--  6—•  '  - m —  i -  + *  120 - -  80--  40--  air—exposure  Time (days)  t  45  3) Ammonium chloride exposure: The best fit linear regression equations, R values and mean initial values ± 2  S.E. of all measured respiratory and internal variables can be seen in Table 4. •  *  There were no significant differences between the initial values of V (t), V (a), 02  T  a m m  02  and plasma [urea] for this experiment and the values for the same parameters  under control conditions. Total 0  2  consumption, V (a), T ^ , and plasma [urea] did not change during oz  NH4CI exposure  (fig 5A), their regression coefficients  were not significandy  different from zero. The regression coefficient for plasma [urea] was significantly different from that derived under control conditions, but the coefficients for V (t), 02  V (a) and T 02  a m m  were not.  Urea excretion represented 9.9% of total nitrogen excretion under control conditions, with an excretion rate of 30.2 ± 8.0 mmol/kg/hr cf 606.6 ± 86.8 mmol/kg/hr total ammonia excretion. Urea excretion rates did not increase during NH4CI exposure.  4) Hypoxic exposure: The effect of aquatic hypoxia without access to air breathing on measured respiratory and internal variables can be seen in Figure 6. At P no variable showed a significant change. At P increased significantly over control values. both  w 0 2  w 0 2  = 85 mmHg, V  C 0 2  = 111 mmHg, and R . E . both  Arterial plasma total C 0  2  and P  a C 0 2  46 Table 4: Best-fit linear regression equations and mean initial values of respiratory, blood gas and excretory variables during 900u.mol/l NH C1 exposure. 4  Variable  N  n  Best-fit  R  v (t)  6  48  y=0.39x+44.47.,  V (a)  6  48  T  7  [urea] Urea exc.  02  G2  •*• amm  R.C.S.E.  Mean±SE  0.01  0.57  50.46+9.95  y=0.20x+7.10  0.01  0.25  8.27±2.88  26  y=6.61x+340.5  0.07  4.80  310.8+38.4  7  26  y=7.10x+112.6  0.15  3.45  108.8±17.7  6  52  y=-0.74x+23.8  0.03  0.57  30.2±17.9  2  N = number of fish, n = number of observations. R.C.S.E. = regression coefficient standard error. Urea exc. = urea excretion rate. Units: V (t), V (a) = mg/kg/hr; T ^ , [urea] = u.mol/1; urea exc. = (xmol/kg/hr. 02  Q2  47  Figure 5A: The relationship between V (t) 02  and V (a) and Time (days) during 900u.mol/l 02  NH4CI exposure. Each symbol represents an individual animal.  V 0 o  2  (a) (mg/kg/hr) o  o  o»-BHB»  • -  (A  o  o  -+-  CD  •  o  • o a  3"  Q  E L O *  i•  ••••  o  V 0  2  (t) (mg/kg/hr)  49  Figure 5B: The relationship between T ^ , [urea] and urea excretion and Time (days) during 900u.mol/l NH4CI exposure. Each symbol represents an individual animal for urea excretion.  50  _>  1000  -r  s3?  750  +  o E  E E D  O  n = 6  500 4 -  250  E CO  o_ \  450+  n  =  100-  n = 6  E 3 o  o  o E 3 c o u  X V D V  7  5  +  50 +  A •  251  A  D  A  -A -  o*10  Time ( d a y s )  12  51  Figure 6: Respiratory and blood gas variables at different levels of aquatic hypoxia. * = significandy different from control at P<0.05.  R.E.  o b  o In  o -+-  -4—  VC02 -t—  10 b  s  - I —  -t—  (mg/kg/hr)  a o  V0  2  (mg/kg/hr)  ro o  M  03  a  -t—  o —1—  O) CO  TJ  3  -  o  -*  3 3 x  00  II CO  3  II  01  CD  id  3  II OJ  plasma T  o o  a  o o  m  m  0*mol/l)  u o o  -t—  P  q  C  0  2  &  o o -f  -t—  (  m  m  Hg)  T  o o  —(-  aC02  (mmol/l)  b -t—  N>  -4-  O  Ul O  1  01 01  "0 O NJ  3  3  II -J  00  3  II cn  IE  CO  01  CO ui ro  dropped significantly.  A l l other variables did not change.  At P  w 0 2  = 59 mmHg,  only three of eight fish survived 24 hours, and blood samples were obtained from only one animal. V  0 2  were not significant. value.  dropped in all three animals, as did V  the mean control value. At P  , but the differences  Respiratory exchange ratio was similar to the mean control  Arterial blood pH was very low, as were T  control value.  C 0 2  w 0 2  a C 0 2  and P  aC02  , as compared to  Arterial plasma total ammonia was similar to the mean = 30 mmHg, none of the fish survived more than two  hours of hypoxic exposure.  54 DISCUSSION A i r Exposure: A number of actinopterygian fish are known to be capable of surviving periods of water deprivation, e.g Symbranchus marmoratus (Bicudo and Johansen 1979) and Lepidogalaxias  salamandroides  (Pusey  1986).  This capacity must involve  the ability to avoid desiccation, depletion of energy stores, and toxic accumulation of wastes.  No studies to date have investigated  the physiological changes  associated with water deprivation in the above animals, although each is able to breathe both water and air.  There are, however, a number of studies of the  responses to water deprivation seen in other bimodally breathing vertebrates, responses commonly described as "aestivation". The african lungfish, Protopterus surrounding itself  in a mucus  sp. burrows during periods of drought,  cocoon  (Smith  1961). Various anurans,  e.g.  Scaphiophus  couchi (Seymour 1973), Bufo marinus, (Boutilier et al. 1979) and  Pyxicephalus  adspersus, (Loveridge and Withers 1981) also burrow in response  to drought. This reduces evaporative water loss.  Oxygen consumption is reduced  in all of these animals, indicating a reduction in metabolism or the use of alternative, anaerobic metabolic pathways.  Protopterus is ammonotelic when in  water, but during aestivation converts all nitrogenous waste to urea (Janssens 1964;  Janssens  and Cohen 1968,  a,b) thereby avoiding the toxic effects of  excessive ammonia accumulation in the tissues.  Urea levels in the blood rise, as  urine volumes decrease to conserve water (Delaney et al. 1977; Babikker and E l Hakeem 1979). A plasma respiratory acidosis develops in aestivating Protopterus  55 (Delaney et al. 1977) and Pyxicephalus adspersi (Loveridge and Withers 1981). In Bufo marinus, a respiratory acidosis develops,  but is gradually corrected  (Boutilier et al. 1979). The acidosis is probably a result of impeded gas exchange across the skin whilst in a burrow, exacerbated in Protopterus by the increased respiratory dead space that results from breathing through a mucus tube extending to the surface of the mud (Delaney et al. 1974). In the present study, northern, cold water adapted Amia calva did not make any of the physiological adjustments to air exposure seen in lungfish and anurans, with no evidence of a metabolic suppression or detoxification of ammonia to urea.  In teleosts, in general, 45 to 100% of total ammonia excretion is by passive diffusion of N H  3  (Randall and Wright 1987).  In this study, prior to air exposure,  plasma [NH ] increases were correlated with water  [NH ] increases.  3  3  Following  air exposure, there were marked increases in plasma [total ammonia] and no clear correlation between water and plasma [NH ] levels. 3  Water [NH ] levels continued 3  to rise, indicating some continued ammonia excretion. In Amia, under aquatic conditions, over 90% of C 0 excretion occurs at the 2  gills (Randall et al. 1981).  During gradual emersion,  there was no respiratory acidosis.  T  a C Q  2  increased, although  Amia satisfied all their oxygen requirements in  air using their respiratory swimbladder, and there was no evidence of a metabolic acidosis.  The rigid, seive like structure of the gills (Daxboeck et al.  probably allowed some continued ammonia and C 0  2  1981)  excretion following air  exposure, by trapping water in the pores between secondary lamellae.  56  Ammonium chloride exposure: A n increase in water [total ammonia] leads to an increase in urea production via uricolysis in the goldfish, Carassius auratus, (Olson and Fromm  1971) and  via ureagenesis in the primitive air breathing fish, Heteropneustes fossilis, and Ratha 1987).  (Saha  In this study, the water [total ammonia] was over two times  that used by Olson and Fromm (1971), but because of the low water p H , (6.5) the [NH ] 3  was  only  1.9  (imols/1; 28% of the mean plasma [NH ]. 3  membranes are relatively impermeable to N H there was  no significant  increase  + 4  (Randall and Wright 1987), so  in plasma [total  exposure, despite high NrL, levels in the water. +  Biological  ammonia] during NH4CI  Amia relied on urea excretion to  remove only 10% of nitrogenous waste under control conditions, and showed no increase in plasma urea or in urea excretion following ten days of NH4CI exposure.  Mommsen and Walsh (1989) report that Amia does not have functional  levels of ornithine cycle enzymes in isolated hepatocytes, unlike a number of aquatic animals that are known to aestivate (Janssens 1964; Saha and Ratha 1987).  There is some evidence that increases in water-borne irritants can cause an increase in air breathing in gar, Lepisosteus osseus, (Smatresk showed no change in V (t) or V (a) during NH C1 exposure. 02  02  4  1988).  Amia  Thus, under  conditions of drought, if an Amia were trapped in a gradually evaporating puddle, a build up of water ammonia levels per se would not lead to an increase in urea production and excretion, or to increased reliance on air-breathing.  57  Hypoxic exposure: Amia are facultative air breathers, and at low water temperatures, the gills are the main site of gas exchange (Johansen et ah 1970). Canada, they overwinter under ice cover.  In Southern Ontario,  As temperature rises, aerial uptake  begins to predominate, but Amia do not die if denied access to air at 30°C under aquatic normoxia (Johansen et al. 1970; Randall et al. 1981).  During acute  aquatic hypoxia, Amia is capable of meeting all of its oxygen requirements by airbreathing (Randall et al. 1981). The present study showed that if denied access to air, Amia  were not capable of sustaining oxygen delivery with the gills at  relatively moderate degrees of aquatic hypoxia.  At P  w 0 2  = 85 mmHg, there were  clear indications of gill hyperventilation, resulting in very low significant increase in V  C 0 2  P  a C 0  and R . E . over control values.  2  levels, and a  This respiratory  alkalosis was presumably offset by a metabolic acidosis, as p H values were not a  significantly different from control values. reduction in V  Q 2  At P  w D 2  = 59 mmHg there was a  , indicating an inability to sustain oxygen delivery via the gills,  and there was only 50% survival after 24 hours at this level of hypoxia. Rainbow trout (Oncorhynchus mykiss), fish adapted to well-oxygenated fast flowing waters, are able to survive at P  w 0 2  = 25 mmHg, at 15°C (Claireaux et al. 1988), and  display a reduction in total metabolism at P  w 0 2  = 80 mmHg and below (Boutilier  et al. 1988). Sturgeon (Acipenser transmontanus) reduce V reduction in aquatic P  Q 2  Q 2  in concert with a  (Burggren and Randall 1978). Amia are clearly incapable  of initiating a reduction in aerobic or total metabolism in response to hypoxia.  58  In summary, these results suggest that northern, cold adapted Amia calva are not able to aestivate, as they are incapable of reducing aerobic metabolism during air exposure, and are not able to detoxify their nitrogenous wastes as urea. Under the conditions of these experiments, air exposure resulted in death of Amia. respiratory swimbladder functions  only to  sustain aerobic metabolism under  aquatic conditions of raised temperature or lowered P exchange during prolonged emersion. of  "aestivating" Amia  Their  w 0 2  , not to aid in gas-  Previous reports (Dence 1933; Neill 1950)  were probably animals that had recently become air  exposed, although it is possible that Amia from the southern areas of the species' range may be capable of aestivation.  59  Chapter 2: Ventilatory and Cardiovascular Responses to Blood pH, Plasma P  C02  , Blood 0  2  content and Catecholamines in Amia  60 INTRODUCTION The results of Chapter 1 indicate that Amia with the added ability to breathe air. Amia, intermediate stage in  the  evolution  is an entirely aquatic animal, but  therefore, is an extant example of an  from  ventilatory control systems in vertebrates.  water-breathing  to  air-breathing  The extent to which ventilatory  responses in Amia are similar to those of water or air breathers is unknown. In  water-breathing fish,  0  2  is  the  primary  stimulus  for ventilatory and  cardiovascular reflex responses (Dejours, 1973; Randall and Jones, 1973; Smith and Jones, 1982; Randall, 1982).  Apart from a modest sensitivity to P  a C 0 2  and/or  p H that has been demonstrated in hyperoxic dogfish (Heisler, Toews and Holeton, a  1988), ventilatory responses by water-breathing fish to changes in plasma P  and  c o 2  blood p H only occur when they are associated with reductions in blood  0  2  content, via Bohr and Root effects (Smith and Jones, 1982; Perry, Kinkead, Gallaugher and Randall, 1989).  Air-breathers (amphibians, reptiles, birds and  mammals) exhibit direct ventilatory and cardiovascular responses to pH , as well as blood 0 a  2  status (Dempsey  and Forster, 1982;  P  a C 0  2  O'Regan and  Majcherczyk, 1982; Scheid and Piiper, 1986; Smatresk, 1990, for reviews). still unclear whether reflex responses in mammals are to P  c o 2  and/or  It is  or pH, as there is  evidence of sensitivity to both (Shams, 1985). These differences between vertebrate groups in the reflex control of breathing are considered to be related to the differential capacitances of water and air for 0 and C 0  2  (Dejours, 1981).  sensitivity to P  C02  2  It is unknown when ventilatory and cardiovascular  / p H first appeared in the evolution of air-breathing. Amia  shows  61 ventilatory sensitivity to Oy, increasing gill ventilation and air-breathing in aquatic hypoxia (Johansen et al, 1970; Randall et al, 1981) but it is unknown whether it also responds to C 0 and/or pH. 2  There is recent evidence that release of circulating catecholamines (NE and E) from chromaffin tissue may mediate ventilatory responses to hypercapnia and acidosis in water-breathing fish (Perry et al, and Randall, 1990).  1989; Aota, Holmgren, Gallaugher  Catecholamines stimulate ventilation in some water-breathing  fish (Peyraud-Waitzennegger, 1979) and air-breathing vertebrates (Dempsey, Olson and Skatrud, 1986), and they are released into the circulation in response to blood acidosis in water-breathing fish (Boutilier, Iwama and Randall, 1986; Tang and Boutilier, 1988; Perry et al,  1989; Aota et al,  1990).  The effects of circulating  catecholamines on ventilation and their potential role in ventilatory responses have not been examined in air-breathing fish. This study compared cardiovascular and ventilatory responses, and endogenous catecholamine release, in Amia exposed to blood acidosis, to transient increases in plasma  P  C 0  2  without acidosis, and to blood acidosis when C  a 0 2  was maintained  above normoxic levels, to discover whether reflex responses were best correlated with P  aC02  , p H or C a  a 0 2  .  Any associated changes in blood catecholamine levels  were recorded and cardiovascular and ventilatory responses to pharmacological doses  of  catecholamines  were  assessed,  under  normoxia  and hypoxia,  investigate their possible role in ventilatory responses to acidosis.  to  62  MATERIALS AND METHODS  Experimental Animals: Bowfin were maintained and temperature acclimated as described in general materials and methods.  Surgical Procedures: Animals were anaesthetized in a buffered (NaHC0 ) tricainemethanesulphonate 3  (MS222) solution at a concentration of 1:10,000 and transferred to an operating table, where they were ventilated with a MS222 solution at 1:20,000.  A dorsal  aortic cannula (PE50, Intramedic) was implanted using the technique of Soivio, Westman and Nyholm (1972).  A n opercular cannula was fitted, using flared  PE190 (Intramedic) passed through a small hole drilled in the operculum, secured with a cuff and sutures.  The fish was allowed to recover in a black plexiglass  box (volume 9 1) with access to a forward space, for airbreathing (volume 1.6 1), for 48 hours before use in an experiment.  D A cannulae were flushed with  heparinized Cortland's saline (Wolf, 1963) twice daily.  Cardiovascular and Ventilatory Measurements: During experiments, dorsal aortic peak systolic blood pressure (P , cmH 0) DA  2  and heart rate (f , beats/min) were measured using a Statham (P23Db) pressure h  transducer attached to the 20 cm, saline-filled dorsal aortic cannula.  Gill  ventilation frequency (f , beats/min) and opercular pressure amplitude (P , cm g  op  H 0) were measured using a Statham (P23BB) pressure transducer attached to the 2  20 cm, water-filled opercular cannula. The output from both transducers was  63 displayed  on  a pen  recorder (Gould  8188-2202-XX).  amplitude was used as an index of ventilatory effort.  Opercular  pressure  The frequency of air  breathing (f ) was visible as large pressure excursions on the opercular trace, ab  associated with changes in f  and P  h  D A  (fig 7).  These air breaths were verified  visually through a small hole in a screen between the experimenter and the bowfin.  Ventilatory and cardiovascular variables were considered to be in steady  state when they remained stable for 30 minutes. Experimental Protocols: Once ventilatory and cardiovascular variables were in steady-state, bowfin were exposed to the following treatments: Series 1: Treatment 1) 2.5 ml/kg Cortland's saline infusion into the D A , followed by one hour recovery, and then 2.5 ml/kg 0.1M hydrochloric acid ( H Q ) infusion, in a Cortland's saline vehicle. Treatment 2) 2.5  ml/kg Cortland's saline infusion, followed by a one hour  recovery period, and then 2.5 ml/kg 0.2M ammonium bicarbonate ( N H H C 0 ) 4  3  infusion, in a Cortland's saline vehicle. Treatment 3) One hour's exposure to aquatic hyperoxia (P created  by  bubbling  100%  0  2  counter-current  to  w02  = 643 ± 12 mmHg),  water  flow  through  a  gas-exchange column, followed by the same infusion series as in treatment 1. Series 2: Treatment A) 0.5 ml/kg Cortland's saline injection, followed by a 2 hour recovery period.  Then, 0.5 ml/kg 10" M epinephrine hydrochloride (Sigma) injection, in a 5  64 saline vehicle, followed by a two hour recovery period. Subsequently, a 0.5 ml/kg 10" M norepinephrine bitartrate (Sigma) injection, in a saline vehicle. 5  epinephrine (E) and norepinephrine (NE) solutions were at pH 7.7.  Both  In half the  animals studied, the order of epinephrine and norepinephrine injections  was  reversed. Treatment B) Three animals were exposed, for two hours, to moderate aquatic hypoxia ( P  w02  = 59+1.9 mmHg), obtained by bubbling N counter-current to water 2  flow through a gas-exchange column, and then treated to E and N E injections as described for treatment (A).  No cardiovascular responses were measured in this  treatment All infusions were performed over 7 to 10 minutes, at approximately 0.3 ml.min" . 1  This infusion rate avoided any struggling associated with irritant or  behavioural responses.  Injections in Series 2 were performed over 1 minute. A l l  animals were used in more than one treatment, assigned randomly, with a 48 hour recovery period between each treatment. Baseline measurements of P , f , f and P DA  h  g  op  were recorded for 10 minutes as a  control, and for 30 minutes post-infusion in Series 1. measured continuously for 1 hour post-injection.  f  ab  In Series 2, variables were  was measured for 30 minutes  post-infusion in Series 1; for 1 hour post-injection in Series 2.  At 5 minutes  post-infusion (or injection), a 1 ml blood sample was withdrawn in both series of treatments. Sample Analysis: 0.5 ml of blood was immediately centrifuged, the plasma decanted and frozen  65 in liquid nitrogen for subsequent analysis of plasma catecholamine levels, as described in general materials and methods.  The remaining blood was analysed  for pH , a  T co2» Paco2> Qo a  a n 2  d P 2» as described in general materials and methods. a0  Data analysis and statistics: Heart rate and f were assessed by counting for 30 seconds within each minute, g  for two minutes immediately prior to intervention, and at 1, 2.5, 4, 10, 15, 20, and 30 minutes following intervention (for Series 2, also at 60 minutes).  P  D A  and P  op  were averaged from 6 measurements taken within the same periods used for measuring f and f . h  g  Cardiovascular and ventilatory responses were normalized for  each time interval as per cent change from control values. transformation, responses were analysed by A N O V A .  Following arc-sine  Within each treatment mean  values of blood gas variables following saline infusion (injection) were compared with mean values following experimental infusions (injection) using a paired t-test. Air-breath frequency following saline and experimental infusions (injection) within each treatment was compared with a paired t-test. Mean values of blood variables during hyperoxia were compared with the same parameters during normoxia using unpaired t-tests. P = 0.05 was taken as the fiducial limit of significance.  66 RESULTS SERIES 1 For all treatments, infusion of 2.5 ml/kg Cortland's saline had no significant effect on steady-state cardiovascular (f  h  and P  D A  ) , or ventilatory (f , P g  and f )  OP  ab  variables (figs 8, 9 and 10).  In Treatment 1, HC1 infusion caused a significant increase in PDA» (figs 7 and 8, table 5).  The changes in P  O P  of infusion, and peak response in both P minutes post-infusion (p.i.). and P  OP  at 10 minutes.  on f  h  or f , g  0  ab  were initiated towards the last minute  D A  and P  OP  occurred between 1 and 5  Blood pressure returned to control at 30 minutes p.i.,  Air-breaths all occurred within the first ten minutes p.i.,  and the majority occurred within the first five minutes. effect  and f  P p>  although some change  There was no significant  is visible in figure  8.  These  cardiovascular and ventilatory changes were associated, at five minutes p.i., with a significant decrease in p H , T a  a C 0 2  and C  a 0 2  , and a significant increase in P o2> P o2 aC  a  and the concentrations ([NE] and [E]) of circulating catecholamines (table 5).  In Treatment 2, N H H C 0 infusion had no significant effects on P 4  or f  ab  (fig 9, table 5).  3  D A  , f, P h  OP  f  g  At five minutes p.i. there was no significant change in pH,,,  C 2» P o2 or [NE] and [E] as compared with values obtained following saline a0  a  infusion, but T  a C 0 2  and P  A C 0 2  showed a significant increase (table 5).  67  Table 5: Effects of HC1, N H H C 0 and HC1 in Hyperoxia on f , blood gases, [NE] and [ E ] . 4  3  ab  HC1  NH4HCO3 exp.  HC1 + Hyperoxia sal. exp.  sal.  exp.  sal.  0.32 ±0.36  4.34* ±1.48  0.66 ±0.46  1.00 ±0.74  0  0  7.60 ±0.04  7.31* ±0.07  7.66 ±0.02  7.67 ±0.03  7.67 ±0.03  7.31* ±0.10  9.47 ±0.12  8.65* ±0.19  9.58 ±0.10  12.41* ±1.31  10.15 ±0.21  9.05* ±0.15  PaC02  8.80 ±0.84  15.29* ±1.68  7.39 ±0.30  9.61* ±0.66  8.35 ±0.80  15.1* ±2.65  Pa02  35 ±5  48* ±8  59 ±18  51 ±9  368+ ±29  313+ ±51  5.8 ±0.7  4.1* ±0.6  6.0 ±0.8  5.4 ±0.6  10.2+ ±0.2  8.5+* ±0.7  [NE]  13.3 ±5.5  720.0* ±240.0  14.2 ±5.2  17.4 ±7.2  57.2 ±28.0  48.0 ±12.0  [E]  9.0 ±2.2  703.1* ±194.0  21.0 ±3.3  16.2 ±2.4  54.4 ±27.8  47.1 ±12.2  fab  pH  a  TaC02  Co a  2  -  -  Values = mean ± S.E., N = 6 * = significantly different from control; + = significantly different from normoxic control (P=0.05) Units: f = breaths/hr; T = mmolA; P and P = mmHg; C = vol.%; [NE] and [E] = nmol/1 ab  a C 0 2  a C 0 2  a 0 2  a 0 2  68  Figure 7: Representative traces of blood pressure and ventilation. A) A n air breath (ab).  B) The effects of HC1  infusion during aquatic normoxia, (ab = air breath). C) The effects of H Q infusion during aquatic hyperoxia. inj.= infusion.  40r  69  30 O  20  CM  E  10  ab  L  o 1  [ I  VVVVVVVVVVVvVVV^WVVV^^  op mm  40 30 O  DA  20  CM  E  B  10  o HIHIIl|V|ll|IIHII|llfTlH n  IMII'IIIIMI  D  op  40 30  o  CM  DA  20 •  X  E  10 •  o  1  mmMmmmm control  I  Inj.  mmmmmmmmmmi MUHmmm vlflmmmtm 1  2 TIME Imins)  15  30  P  70  Figure 8: Mean % change (± S.E.) in P A» D  Pop and f  following H Q infusion, n = 6. C = control, shaded bar represents infusion period.  s  % c h a n g e in f  % c h a n g e in P  % c h a n g e in  % c h a n g e in P Q ^  Figure 9: Mean per cent change (± S.E.) in P , f , P DA  h  op  and  following NH4HCO3 infusion, n = 6. C = control, shaded bar represents infusion period.  % c h a n g e in f  % c h a n g e in P  % c h a n g e in  % c h a n g e in  P Q ^  74  In Treatment 3, aquatic hyperoxia caused a significant increase in P as compared with normoxic conditions (Table 5). ventilation (f  g  and C  a 0 2  ,  There was no reduction in gill  or P ) as compared with fish in normoxia, but there was no air op  breathing (table 5). in  aOZ  HC1 infusion during hyperoxia effected no significant changes  cardiovascular or ventilatory  variables, except P , which was DA  increased  immediately p.i., and remained elevated until 20 minutes (Figures 7 and 10). There is no evidence of the response profile for P infusion in normoxia. C 2, a 0  and T  a C 0 2  and f visible following acid  op  g  At five minutes p.i, there was a significant drop in pH,,,  , and a significant increase in P  [NE] and [E] did not change (table 5).  a C 0 2  .  Blood 0  Arterial blood 0  2  2  partial pressure, content was still  significantly higher than normoxic values five minutes following HC1 infusion (table 5).  SERIES 2 : Saline injection resulted in no change in steady-state values of P , f , P , f DA  (figs 11 and 12) or f  ab  h  op  s  (table 6).  Treatment A) Resting plasma [NE] and [E] measured in this study are an order of magnitude higher than those reported for water breathing fish (Perry et al. 1989),  75  Figure 10: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f  following HC1 infusion during hyperoxia. n = 6 C = control, shaded bar represents infusion period.  s  % c h a n g e in f  % c h a n g e in P  % c h a n g e in  % change in P p ^  77 and injection of N E and E stimulated a large endogenous release, with both [NE] and  [E] increasing after either N E or E infusion (table 6).  Norepinephrine  injection stimulated significant cardiovascular and gill ventilatory responses; P , DA  P  op  and f all increased (fig. 11), although f did not. g  h  P  D A  increased immediately,  and remained significantly elevated until 4 mins p.i., P  op  2 mins p.i. and returned to control levels at 10 mins.  f increased at 2.5 minutes  p.i.,  and returned to control at 10 minutes.  increased significantly at  g  There was no stimulation of air  breathing (table 6).  There was no significant change in p H , but a significant  increase in P  a 0 2  a 0 2  and C  a  (table 6).  Following epinephrine injection, f , P h  all increased significantly, but f and f g  ab  D A  and P  did not change (fig. 11, table 6). P  D A  op  rose  immediately, and remained elevated until 15 mins post-injection, and f increased h  at 2 mins and returned to control levels at 15 mins post-injection.  P  2 mins and returned to control levels at 15 mins post-injection. effected no significant change in p H or P a  6).  a02  op  increased at Epinephrine  , but significantly increased C  Epinephrine appeared to stimulate cardiovascular  a 0 2  (table  variables more than  norepinephrine, which had a greater effect on ventilation, but these differences were  not  significant  when  compared at each  time  interval with  Catecholamine infusions at doses of 1 ml/kg 10" M or 10 4  3  a t-test.  M , during aquatic  normoxia, did not stimulate air breathing. Treatment B)  During moderate hypoxia, there was a significant increase in  pre-injection f , but no change in gill ventilatory variables or blood gases, as ab  compared with normoxia (table 6).  Opercular pressure amplitude and f were not g  78  Table 6: Effects of N E and E on f and blood gases in normoxia and hypoxia, and on [NE] and [E] in normoxia. ab  Saline  NE  E  0.32+0.36  0.32+0.36  0  6.67+1.33+  6.00+0+  6.00±2.00+  7.65±0.03  7.68±0.01  7.67±0.03  7.73±0.07  7.70±0.05  7.77±0.06  43±8  72±13*  53±7  41±7  48±8  38±7  5.8±0.4  7.2±0.5*  6.8±0.5*  5.8+1.1  7.1±1.0*  5.8±0.2  Normoxic [NE] 21.1+5.0  564.8+90.0*  416.2±183*  Normoxic [E]  231.0+88.1*  1079+160*  Normoxic f Hypoxic f  ab  ab  Normoxic p H Hypoxic p H  a  Normoxic P Hypoxic P  a 0 2  a 0 2  Normoxic C Hypoxic C  a  a 0 2  a 0 2  11.1±3.3  Values = mean ± S.E.; N = 6 in normoxia; 3 in hypoxia. * = significantly different from saline, + = significantly different from normoxia (P=0.05) Units: f = breaths/hr; P = mmHg; C = vol.%; [NE] and [E] = nmol/1. ab  a 0 2  a 0 2  79  Figure 11: Mean per cent change (+ S.E.) in P , f , P DA  h  op  and f  following N E or E injection during normoxia. n = 6. C = control, shaded bar represents infusion period.  {  Figure 12: Mean per cent change (± S.E.) in P  op  and fj  following N E or E infusion during moderate hypoxia, n C = control, shaded bar represents infusion period.  % c h a n g e in f  % c h a n g e in P  83 significantly higher than normoxic levels, but this may reflect the small number of fish studied, and the fact that the same fish were not measured under both conditions.  N E and E had no significant effect on P  increase f  (table 6).  ab  op  and f (fig 12) and did not g  Norepinephrine effected a significant increase in  there were no other significant effects on blood gases.  C 2» a Q  but  84  DISCUSSION SERIES 1. Cardiovascular and ventilatory responses seen in treatments 1, 2 and 3 have been correlated with P  aC02  , p H and C a  a 0 2  levels measured at five minutes p.i., to  assess the possible role of each of these blood gas variables in the reflex responses observed.  Possible mechanisms behind observed responses will be  discussed.  The relationship between P  aC02  and cardiovascular and ventilatory responses:  Acid infusion in normoxia stimulated P , P DA  with a significant increase in P  aC02  caused a significant increase in P  .  op  and f , and was associated ab  Ammonium bicarbonate infusion, however,  a C 0 2  (table 5) that was not associated with any  significant changes in f , PDA» Pop> fg or f . h  ab  Acid infusion in hyperoxia also  increased P o2> hut there was no stimulation of ventilatory variables.  These  aC  results indicate that Amia  do not show cardiovascular or ventilatory sensitivity to  an increase in P 2aC0  The relationship between p H and cardiovascular and ventilatory responses: a  Acid infusion in normoxia was associated with a significant reduction in p H , a  and significant increases in P , P DA  op  and f . ab  During hyperoxia, acid infusion  resulted in a significant reduction in p H that was associated with an increase in a  P , but no ventilatory responses. DA  This suggests that a reduction in p H per se  causes increases in blood pressure, but not gill ventilation or airbreathing.  a  85  The relationship between C  a 0 2  and cardiovascular and ventilatory responses:  Acid infusion in normoxia was associated with a significant reduction in C and elicited increases in P , P DA  significant decrease in C  a 0 2  op  and f . ab  ,  Acid infusion in hyperoxia caused a  , but at 5 minutes p.i. C  than control, normoxic, levels.  a 0 2  a 0 2  was still significantly higher  There were no ventilatory responses in hyperoxia.  This suggests that both gill ventilation and air-breathing are stimulated by a reduction in C  a 0 2  below normoxic levels in Amia.  These results indicate that the primary stimulus for ventilatory responses to internal acid-base disturbances in Amia is hypoxaemia, as is the case for waterbreathing fish, and that Amia do not show a ventilatory response to P o2- If  m  e  r  e  aC  is a ventilatory response  to p H , then it is inhibited by hyperoxia. a  The  cardiovascular responses are interesting, as they indicate that there is an effect of a reduction in p H on blood pressure. a  It is unknown how a reduction in pH,,  might effect increases in P , although thromboxane release associated with acid DA  infusion stimulates increases in blood pressure in mammals (Shams, Peskar and Scheid, 1988). There is evidence for water-breathing fish that ventilatory and cardiovascular responses to changes in external and internal 0  2  status are neurally mediated, by  0 -sensitive chemoreceptors in the gills (Milsom and Brill, 1986; Burleson, 1986; 2  Burleson and Smatresk, 1986; Burleson and Milsom, 1986).  There is similar  evidence in lungfish (P. aethiopicus) (Lahiri, Szidon and Fishman, 1970) and in both spotted gar (Lepisosteus oculatus) and longnose gar (L. osseus) (Smatresk, 1986;  Smatresk, 1987).  The site of putative internal receptors in Amia  is  86 unknown. In longnose gar, internally oriented receptors appear to set the level of hypoxic drive, and external receptors influence the balance between gill ventilation and airbreathing, with central integration of the external and internal afferent input (Smatresk, Burleson and Azizi, 1986). In gar, internal hypoxia will stimulate both air-breathing and gill ventilation responses (Smatresk et al., 1986).  In this study,  both air-breathing and gill ventilation were stimulated by a reduction in C  a 0 2  ,  indicating that Amia are more similar to gar than to Ancistrus, where air-breathing responses are only influenced by external hypoxia (Graham and Baird, 1982).  It  is unknown whether the gill ventilation and air-breathing are stimulated by the same peripheral receptors in Amia. In water-breathing  fish, there is evidence that ventilatory responses to internal  acidosis may be Immorally mediated.  It is known that internal acidosis in  normoxic water-breathing fish is associated with a ventilatory increase and release of N E and E into the circulation (Boutilier et al, 1986; Tang and Boutilier, 1988) and that the catecholamine release is in response to a reduction in C al., 1989; Aota et al., 1990). breathing  fish  a 0 2  (Perry et  Both N E and E stimulate ventilation in water-  (Peyraud-Waitzennegger,  1979),  and  Aota  et  al.,  (1990)  demonstrated that the ventilatory response and catecholamine release following acid infusion are abolished by hyperoxia, and that the (3-adrenergic receptor blocker propranolol abolishes the ventilatory response but not the catecholamine release.  This indicates that N E and E might be responsible for stimulating the  ventilatory response to acid infusion in water-breathing fish.  87 In the present study, increases  in blood [NE] and [E] only occurred in  treatment 1, when there was a reduction in C increase in P  op  and f .  a 0 2  below normoxic levels, and an  Thus the correlation between catecholamine levels and  ab  ventilatory responses seen in water-breathing fish holds true in Amia also.  This  suggests that N E and E might be responsible for mediating the observed increases in P  op  and f . ab  Further evidence for this possibility will be gained by looking at  the ventilatory responses to N E and E infusion in Amia. S E R I E S  2:  Infusion of N E and E had pronounced effects on cardiovascular and gill ventilatory variables during normoxia, but not during moderate hypoxia. was no stimulation of airbreathing in either treatment. release seen  There  The large endogenous  following N E and E injection in normoxia is similar to that seen in  the American eel, Anguilla rostrata (Epple and Nibbio, 1985). Catecholamines are known to stimulate ventilation in water-breathing fish (Peyraud-Waitzennegger, occurs is unknown.  1979), but the mechanism by which this stimulation  Catecholamines can cross the blood:brain barrier in fish  (Nekvasil and Olson, 1986), suggesting that the increases in P N E and E may be a centrally mediated effect.  op  and f following g  In air-breathers (mammals),  catecholamines affect ventilation at both central and peripheral sites (Dempsey et al, 1986). It is conceivable that the lack of an air-breathing response to catecholamines during normoxia was the result of a gating effect.  In Amia, a change in  ventilatory pattern, with increased emphasis on air-breathing, occurs in moderate  88  hypoxia (Johansen et al,  1970; Randall et al,  1981; table 6).  Catecholamines  may exert effects on ventilation at a site that will stimulate either gill ventilation or air-breathing, depending on the prevailing level of hypoxic drive. In normoxia, the emphasis was on gill ventilation, so catecholamine infusion increased P f.  op  and  If the lack of an air-breathing response to N E and E in normoxia was the  g  result of a gating mechanism, then one might expect pharmacological doses of N E and E to stimulate increases in f  ab  during moderate hypoxia (f  increase in moderate hypoxia).  This was not the case, indicating that N E and E  ab  still has scope for  exerted effects on a structure responsible for stimulating gill ventilation alone. is unknown why N E and E did not stimulate P Thus, the results of treatments  op  It  and f in hypoxia. g  (A) and (B) indicate that if endogenous  catecholamines do mediate ventilatory responses to internal acidosis in Amia, then they only mediate gill ventilatory responses. It is clear, however, that their release is an adaptive response, as catecholamine infusion increased C  a 0 2  , indicating that  endogenous release would ameliorate the effects of acid infusion on blood 0 2  carrying capacity.  Catecholamines are known to have this effect during acidosis  in water-breathing fish (Perry and Kinkead, 1989).  In  conclusion, it appears that in Amia,  air-breathing and gill ventilatory  responses to a reduction in p H only occur if there is an associated reduction in a  C  a G 2  .  Arterial blood p H may have a direct effect on P .  stimulates  DA  gill  ventilation  but  not  air-breathing.  Catecholamine infusion  Increases in endogenous  catecholamines may mediate gill ventilatory responses to a reduction in C  a 0 2  , and  89 catecholamine release probably ameliorates the effects of acidosis on C  a 0 2  90  Chapter 3 : Ventilatory and Cardiovascular Responses to Increases in Plasma Total C Q and Total Ammonia in Rainbow Trout and Amia. 2  91 INTRODUCTION In water-breathing fish, blood and water 0  2  status is the primary stimulus for  ventilatory responses (Smith and Jones, 1982; Randall, 1982; Shelton et a/., 1986), and the results of Chapter 2 indicate that this is also true for Amia. evidence,  There is  however, that some water-breathing fish may exhibit a ventilatory  response to increases in T  a C 0 2  (Janssen and Randall, 1975), and mammals are  known to show a ventilatory response to increases in T ^ (Wischer and Kazemi, 1974). Janssen and Randall (1975) showed  that infusion of sodium bicarbonate  (NaHC0 ) stimulated ventilation in rainbow trout. This ventilatory response could 3  be an effect of increases in T  a C 0 2  , Pco2 or HC0 ". a  3  N a H C 0 infusion also caused a 3  significant increase in p H (Janssen and Randall, 1975). a  Thus, the ventilatory  response may have been to changes in other blood variables associated with an alkalosis.  For example ammonia, the major end-product of protein catabolism,  dissociates into ionised (NH ) and un-ionised (NH ) states when in solution. The +  4  3  degree of dissociation in plasma is dependent on blood pH, with N H levels 3  increasing as p H increases.  The N H form is freely permeable to cell membranes. 3  Increases in blood p H might lead to changes in ammonia distribution between tissue compartments, with a gradient from alkalotic extracellular compartments to intracellular compartments at lower pH, e.g. the brain.  Ammonia is known to  stimulate ventilation via a central, intracellular effect in mammals (Wischer and Kazemi, 1974), but ventilatory responses to ammonia have not been examined in fish.  Thus there is evidence to suggest that water-breathing fish may show  92 ventilatory responses to all three major respiratory gases, 0 , C 0 and ammonia. 2  2  It is possible that the ventilatory response to increases in blood T mediated  by  a  release  of  catecholamines  into  circulation.  or  a C 0 2  is  Exogenous  catecholamine infusion is known to stimulate ventilation in eels, Anguilla  anguilla  (Peyraud-Waitzennegger,  comm.).  1979), and in rainbow  trout  (Aota, pers.  Catecholamines are released in response to stress in fish (Nakano and Tomlinson, 1967; Perry at al., 1989), and may mediate the ventilatory response to hypoxaemia (Aota et al, 1990). Cardiovascular changes associated with ventilatory responses to increases in T o2 and aC  have not been described in fish.  The present experiment was designed to determine whether water breathing fish (rainbow trout) show ventilatory and cardiovascular responses to increased T and T  a m m  , and to compare responses with those seen in Amia.  a C 0 2  For trout, an initial  experiment controls for the effects on ventilation of changes in pH , produced by a  sodium hydroxide or hydrochloric acid infusion into the dorsal aorta.  This is  followed by an investigation of the effects on ventilation of changes in T  a C 0 2  and  Tamm* produced by sodium bicarbonate, ammonium bicarbonate and ammonium chloride infusion. T  a m m  The possibility that ventilatory responses to increased T  are stimulated by a reduction in C  catecholamines increases in T  is investigated. a C 0 2  and T  those seen in trout.  a m m  a 0 2  a C 0 2  and  or increases in the levels of circulating  Ventilatory and cardiovascular responses to  are described in Amia, and the results compared with  93 MATERIALS AND METHODS.  Experimental animals: Rainbow trout, Oncorhynchus my kiss, weighing between 240 and 360 g, from the Sun Valley Trout Farm (Mission, B.C.), were maintained in large outdoor tanks, with a constant flow of dechlorinated Vancouver tapwater. regularly with trout chow.  Fish were fed  Water temperature was 8 to 12°C.  Amia (500 to 1100 g) were maintained and temperature acclimated as described in general materials and methods.  Animal Preparation: Two different surgical procedures were followed to prepare trout for ventilatory measurements, but all Amia were treated as in procedure 2:  Procedure 1. Trout were anaesthetized  in a buffered (NaHC0 ) MS-222 solution at a 2  concentration of 1:10,000, and transferred to an operating table where they were maintained at an MS-222 concentration of 1:20,000.  Dorsal aortic cannulae (PE  50) were implanted using the technique of Soivio, Westman and Nyholm (1972). A latex mask was sutured below the eyes and in front of the opercula, and attached to the divider in a Van Dam box.  In this way, all water flow from  anterior to posterior chambers was via the mouth and gills (Cameron and Davis, 1970). The water level was adjusted so that the fish had a positive pressure head  94 across the gills, and the animal allowed 48 hrs to recover.  The D A cannula was  flushed twice daily with heparinised (1:1,000) Cortland's saline (Wolf, 1963).  Procedure 2. Trout or Amia were anaesthetised and fitted with a dorsal aortic cannula as described above.  A n opercular cannula was fitted, using flared P E 190 tubing  passed through a small hole drilled in the operculum, secured with a cuff and sutures.  Trout recovered from surgery in individual black plexiglass boxes, and  Amia in individual plexiglass boxes with access to a forward airspace, to allow air-breathing, for 48 hrs. The D A cannula was flushed as described above.  Ventilatory and cardiovascular measurements: Procedure 1. One hour before the experiments, water level was adjusted in the Van Dam box, so that the trout had to ventilate actively, and any water overflow from the posterior chamber was a result of ventilation. measured  by  collecting  the  overflow  for  Ventilation volume (Vg) was  two  one  minute  periods.  No  cardiovascular measurements were made in fish treated by this procedure.  Procedure 2. Following recovery, the water-filled opercular cannula was presssure transducer (Statham P23BB).  attached to a  This allowed gill ventilation rate  (f , g  beats/min), and opercular pressure amplitude (P , cm H 0 ) to be recorded and op  2  95 displayed on a brush recorder (Gould 8188-2202-XX). of stroke volume.  P  op  was used as an index  In Amia, air-breaths were visible as large pressure excursions  in opercular pressure amplitude, and were verified visually via a small hole in a screen separating experimenter and fish.  In Series 3 and 4 (see below) the saline-  filled D A cannula was attached to a pressure transducer (Statham P23dB).  This  allowed heart rate (f , beats/min) and dorsal aortic blood pressure (P , cm H 0 ) h  DA  2  to be recorded and displayed on the same recorder as used for ventilatory recordings.  Protocol:  Trout were used in three series of infusions.  Series 1: Trout surgically prepared by procedure (1) were exposed to the following treatments: 1) 5 ml/kg Cortland's saline infusion. 2) 5 ml/kg 0.05 M sodium hydroxide (NaOH) infusion, in a Cortiand's saline vehicle.  3) 5 ml/kg 0.05 M hydrochloric acid (HC1) infusion, in a Cortland's saline vehicle.  Series 2: Trout prepared by procedure (2) were exposed to the following treatments: 1) 5 ml/kg 0.2 M sodium chloride (NaCl) infusion, in a Cortland's saline vehicle.  96 2) 5 ml/kg 0.2 M sodium bicarbonate (NaHC0 ) infusion, in a Cortland's saline 3  vehicle. 3) 5 ml/kg 0.2 M ammonium bicarbonate ( N H H C 0 ) infusion, in a Cortland's 4  3  saline vehicle. 4) 5 ml/kg 0.2 M ammonium chloride (NH4CI) infusion, in a Cortland's saline vehicle.  Series 3: Trout prepared by procedure 2 were exposed to NaCl, N a H C 0 , N H H C 0 and 3  4  3  HC1 infusions as described above, and ventilatory and cardiovascular parameters measured.  Amia were used in the following treatments:  Series 4: 1) 5 ml/kg 0.2 M NaCl infusion, in a Cortland's saline vehicle. 2) 5 ml/kg 0.2 M NH^HCO;, infusion, in a Cortland's saline vehicle.  A l l infusions were performed over 7 to 10 minutes, at 0.6 ml/min.  At this  infusion rate, there were no signs of irritant or behavioural responses. Ventilation was measured for a control period before each infusion, and then for 60 minutes following infusion in Series 1 and 2, and for 30 minutes in series 3 and 4.  Heart rate and P  D A  were measured for a control period before infusion  97 and for 30 minutes post-infusion in Series 3 and 4.  Immediately prior to each  infusion, a 1.4 ml blood sample was collected, and replaced with an equal volume of heparinised saline.  At five minutes post-infusion (p.i), another 1.4 ml blood  sample was collected, and replaced with an equal volume of heparinised saline. In Series 1, 2 and 4, 500 ul of blood were immediately centrifuged, the plasma decanted and frozen in liquid nitrogen for subsequent analysis of plasma [NE] and [E], as described in general materials and methods. remainder of the sample was analysed for p H , T a  general materials and methods. ([NH ]) were calculated from T 3  materials and methods. P  aOZ  a C 0 2  a C 0 2  Arterial plasma P and T  amra  In Series 1 and 2, the and T ^ , as described in  a C 0 2  and N H concentrations 3  , respectively, as described in general  In Series 3 and 4, blood was analysed for pH,, C  a 0 2  and  , as described in general materials and methods.  Data Analysis and Statistics: In Series 1, ventilation was measured directly, for two minutes pre-infusion and at designated intervals up to 60 minutes post-infusion . In Series 2 to 4, f  g  was  calculated for designated time intervals by counting 30 seconds in each minute, up to 30 minutes.  P  op  was averaged from six measurements taken within the same  period as used to calculate f . g  In Series 3 and 4, f was measured by counting 30 h  seconds in each minute, at the same intervals counted for f . g  Peak systolic P  was averaged from six measurements taken within the same period as f . h  D A  In all  treatments, individual ventilatory and cardiovascular responses were normalised as % change from control (pre-infusion) values.  Following arc-sine transformation,  98 the significance of responses was assessed by A N O V A . infusion  in  Series  3),  the  variance  of  the  In one case (NaHC0  transformed  ventilatory  3  and  cardiovascular measurements within each time interval was heterogenous, and so a non-parametric, Kruskal-Wallis analysis by ranks was used.  In all treatments,  blood and plasma parameters were compared before and after infusion using a paired T-test. P < 0.05 was taken as the confidence level of probability.  99  RESULTS  Series 1: Saline (control) infusion resulted in no significant changes in V g (fig  13).  NaOH and HC1 infusions both caused significant changes in V g (fig 13).  All  three infusions resulted in significant changes in blood gases (table 7). Following saline infusion, there was a small but significant drop in p H and a  increase in T  and P o2-  a C 0 2  aC  Plasma  and [NH ] did not change (table 7) and 3  neither did [NE] and [E] (table 9). At 5 minutes following NaOH infusion, there was no significant change in ventilation, p H  a  was  significantly  significantly depressed.  higher than control values,  and P  a C 0 2  was  Arterial plasma T 2» X ^ , and [NH ] did not change C0  3  (table 7). The level of circulating catecholamines did not change (table 9).  At 20  minutes p.i., there was a significant increase in ventilation that was sustained until 45 minutes post-injection (fig 13). HC1 infusion produced a different response.  At the end of infusion, there was  an increase in Vg, which remained significantly elevated until 12 minutes postinfusion (fig 13). with a significant significantly, but T  This increase in ventilation was associated, at 5 minutes p.i, drop in p H a m m  a  and T  a C 0 2  .  Arterial plasma P  and [NH ] did not change (table 7).  increased significantly (table 9).  3  C 0 2  increased  Plasma [NE] and [E]  Table 7: Blood Gas Measurements for Series 1 and 2. pH  a  Series 1  PaC02  (mmol)  (mmHg)  T amm (umol)  [NH ] (umol;  T C02 a  3  Pre-Saline  7.86 ±0.02  10.64 ±0.89  3.8 0.2  97.1 ±16.6  1.5 ±0.2  Post-Saline  7.81* ±0.03  11.13* ±0.97  4.5* ±0.1  108.6 ±26.9  1.5 ±0.3  Pre-NaOH  7.80 ±0.01  8.82 ±0.3  3.7 ±0.1  56.7 ±9.1  0.8 ±0.1  Post-NaOH  8.06* ±0.08  9.94 ±0.68  2.2* ±0.4  69.5 ±10.6  2.2 ±0.8  Pre-HCl  7.75 ±0.02  8.84 ±0.59  4.3 ±0.3  66.0 ±6.2  0.8 ±0.1  Post-HCl  7.60* ±0.03  7.34* ±0.49  5.0 ±0.5  65.3 ±3.0  0.6 ±0.1  Pre-NaCl  7.91 ±0.06  9.64 ±0.57  2.7 ±0.2  183.5 ±24.3  2.7 ±1.0  Post-NaCl  7.84* ±0.06  10.21* ±0.56  3.3* ±0.3  233.0 ±27.3  2.9 ±0.9  8.03 ±0.04  10.04 ±0.84  2.1 ±0.1  204.7 ±42.3  3.9 ±1.4  8.16* ±.03  15.66* ±0.93  2.3 ±0.2  205.3 ±39.7  5.3 ±1.4  7.85 ±0.04  10.06 ±0.19  3.2 ±0.2  205.2 ±35.1  2.3 ±0.2  7.85 ±0.03  11.69* ±0.22  3.7* ±0.3  1219.3* ±94.2  14.7* ±1.9  7.86 ±0.03  10.36 ±0.20  3.2 ±0.2  138.0 ±25.3  1.6 ±0.2  Series 2  Pre-NaHC0  3  Post-NaHC0  3  Pre-NH HC0 4  3  Post-NH HC0 4  Pre-NH C1 4  3  101 Post-NH Cl 4  7.52* ±0.05  8.44* ±0.14  5.7* ±0.5  All values = mean ± S.E. * = significantly different from pre-injection (P<0.05).  2685.2* ±543.0  14.3* ±2.0  «  Figure 13: Mean per cent change (± S.E.) in V g following NaCl, NaOH or HC1 infusion, n = 6. C = control, shaded bar represents infusion period.  103  104 Series 2: NaCl infusion did not result in any significant changes in ventilation (fig 14). Plasma [total ammonia] and [NH ] did not change, p H showed a small but 3  significant decrease, and T  and P  a C 0 2  a  aC0  2 a small but consistent increase (table 7).  Catecholamine levels did not change significantly (table 9). NaHC0 , N H H C 0 3  (fig  14),  4  3  and NH4CI all caused significant increases in ventilation  and significant  changes in blood and plasma variables  Representative traces of ventilatory responses to NaCl, NaHC0  3  (table  7).  and NH4HC0  3  infusion are presented in fig 15. NaHC0  infusion increased both P  3  within 12 minutes p.i. and f  g  op  and f . g  P  returned to control values  op  within 8 minutes p.i. (figs. 14 and 15).  These  ventilatory responses were associated, at 5 minutes p.i., with a significant increase in p H and T a  a C 0 2  .  Arterial plasma P  C02  , T ^ and [NH ] did not change (table 7). 3  The level of circulating catecholamines increased significantly (table 9). NH4HC0  3  infusion resulted in an immediate increase in P , which remained op  significantly elevated until 20 minutes post-infusion. however, did not change (figs 14 and 15). significant increase in T  a C 0 2  , P  aC02  Gill ventilation frequency,  At 5 minutes p.i., there was a  , T ^ and [NH ], but no change in p H (table 7). 3  a  Neither [NE] nor [E] increased significantly (table 9). NH4CI infusion stimulated both P  op  and f . g  Opercular pressure amplitude  increased immediately, and remained elevated until 3 minutes p.i., but f did not g  Figure 14: Mean per cent change (± S.E.) in P  op  and f  following N a H C 0 , N I ^ H C O , , HC1 and NaCl 3  infusion, n = 6. C = control, shaded bar represents infusion period  g  % change  % change  % change  % change  Figure 15: Representative traces of ventilatory responses to N a H C 0 , N H H C 0 , HC1 3  4  3  and NaCl in rainbow trout, and N H , H C 0 and NaCl 3  in Amia.  Rainbow trout  108  2i  NaHCO,  op  cmH  NH HCQ 4  • op  cmH,  1 min HCI op  cmH.  NaCl  2  op  cmH Control  1  2.5  infusion  15  30  Time (mins)  Amia NH HC0 4  cmH 0 2  |  1 min 2.—  NaCl  op  cmH,0  -— •— T . Control infusion  1  —  2.5 Time (mins)  15  •  30  3  3  109  increase until 3 minutes p.i., and returned to control levels at 8 minutes postinfusion (fig 14).  These ventilatory changes were associated with a significant  decrease in p H and T a  a C 0 2  , and an increase in P  aC02  , T ^ , and [NH ] (table 7). 3  There was no significant increase in [NE] and [E], but resting levels were unusually high in all animals in this treatment, and the data were not included in Table 9.  Series 3: NaCl infusion had no significant effect on either ventilatory variables or f , but h  caused a significant increase in P in pH , P a  a 0 2  or C  a D 2  D A  (fig 16).  NaCl caused no significant changes  (table 8), or [NE] and [E] (table 9).  N a H C 0 , N H H C 0 and 3  4  3  HC1 stimulated both cardiovascular and ventilatory variables (figs 17, 18 and 19). A representative trace of the effects of HC1 infusion on ventilation is presented in fig 15. N a H C 0 infusion resulted in a significant increase in P , f and P 3  17).  op  g  D A  and f (fig h  In two fish, ventilatory increases were up to 500 per cent of pre-injection  values.  These responses were associated with a significant increase in p H , a a  significant decrease in P02» but C a  a 0 2  did not change (table 8).  NHjHCO^ infusion elicited a significant increase in P , f , P op  There were no significant changes in pH , C a  a 0 2  or P  aQ2  g  D A  and f (fig 18). h  (table 8).  HC1 infusion elicited a significant increase in P , f and P , but there was no op  significant change in f (fig 19). h  g  DA  At 5 minutes p.i., these ventilatory responses  Table 8: Blood Gas Measurements for Series 3 and 4 pH  a  C 02 a  Pa02  (vol.%)  (mmHg)  Series 3 : Trout Pre-NaCl  7.77 ±0.02  7.5 ± 0 . 6  147 ±8  Post-NaCl  7.75 ±0.02  6.5 ±0.3  142 ± 4  7.76 ±0.03  7.4 ± 0 . 8  135 ± 6  8.74 ± 0 . 1 0 *  6.4 ± 0 . 9  80 ± 1 1 *  7.85 ±0.01  7.6 ± 0 . 6  143 ±8  7.81 ±0.02  7.5 ±0.7  145 ±3  Pre-HCl  7.77 ± 0 . 0 2  6.6 ±0.8  140 ± 7  Post-HCl  7.51 ± 0 . 0 5 *  5.5 ± 0 . 8 *  196 ± 2 7  Pre-NaCl  7.68 ±0.03  6.7 ± 1 . 2  48 ±9  Post-NaCl  7.69 ±0.03  5.4 ± 1 . 2 *  42 ±6  7.72 ±0.01  6.4 ± 0 . 6  30 ±6  7.63 ± 0 . 0 3 *  5.4 ±1.1  27 ±5  Pre-NaHC0  3  Post-NaHC0  3  Pre-NH HC0 4  3  Post-NH HC0 4  Series 4 :  Amia  Pre-NH HC0 4  3  Post-NH HC0 4  3  3  A l l values = mean ± S.E.; n = 6 in Series 3, n = 5 in Series 4, * = significantly different from pre-infusion (P<0.05).  Table 9: Plasma [NE] and [E] [NE] Pre  [E] Post  Pre  Post  Trout i  NaOH  11.7+4.72  3.19±0.62  1.72±0.56  1.10±0.29  HC1  1.27±0.48  6.17±0.71*  3.67±0.70  32.4±11.0*  NaHC0 3.85±1.12  22.8±6.65*  4.05±1.36  61.3+21.3*  NH4HGO4.ntO.8O  6.96±1.38  12.7+3.47  39.6±17.2  NaCl  3.60±0.42  3.32±0.57  10.7±3.27  11.4±2.01  Saline  10.7±3.23  8.03+1.87  5.41+1.57  3.43±1.48  NH4HCOl8.4il.94  31.7+6.99  16.3±3.65  27.9±7.32  NaCl  23.1±8.01  7.64±2.20  23.4±13.3  3  Amia  13.4±4.57  Units = n M All values = mean ± S.E., * = significantly different from pre-injection.  112  Figure 16: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f  following NaCl infusion in rainbow trout, n = 6. C = control, shaded bar represents infusion period.  £  % change in fg  I  o  I  •••••••••••••#••••••••#( • • •••••••••••••••••••• • • • • ••••••••#»•••••#*•• • •* • •••••••••••••••••#< i • » • • »••••••••«•••••*•••«#•• •••«*«ittt«*«at«a« •••• <  CD + Z5 CO  ho  o to cn  0  % change in P p % change in f 0  n  % change in Pp^  H  1-  114  Figure 17: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f  following N a H C 0 infusion in rainbow trout, n = 6. 3  C = control, shaded bar represents infusion period.  £  % change in fg O o  o  Oi o  H  o o  % change in P p % change in f 0  -•  o o  •••••••••••ft*******  f KH CD  ZJ  to o  ro  o ^  (o  + +  h  •• •• ••• • •  • • • • »•••••>•••••••••••••••• • • • » » • • • • • t •••••••••••!••••••••••* *••••< » • • • ••>•• • • # • • • • # • • • • • • « •••••• t ••••*  —  n  % change in P Q A .  Figure 18: Mean per cent change (± S.E.) in P , f , P DA  h  op  and  following N H H C 0 infusion in rainbow trout, n = 6 4  3  C = control, shaded bar represents infusion period.  % change in f I  cn  o  cn  ro o  cn  o  —* O  o  [...I....'....  —* Cn  o  % change in P  q  K3 O  o  -*  oO  ....  o p  % change in  % change in Pp^  118  Figure 19: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f  following HC1 infusion in rainbow trout, n = 6 C = control, shaded bar represents infusion period.  {  %  c h a n g e  -» o o  in fg  % change  — ro o> o o o  -»  in P  -•  Q  p  % change  M  -»  in f  -»  n  % change  M  O MIIUIIIIMIIHIIIMM  I M I I I t t l l t t l l M M I I U I  •••••••••*••»••••*••*• »• • •• • i• •* • •••••••**•< • •••••*••••••••••*•••••  ••  •••*••••••••••••  • ••• x* ••••••••••••••  1 •  •  - " '  ?  I 3  i >  l-CH  CD  ZJ 0) O '  cn •  O '  I-OH  MD-i  i nP Q ^  120 were associated with a significant reduction in p H and C a  change in P  a 0 2  aQ2  , but no significant  (table 8).  Series 4 : Gill ventilation frequency under control conditions was significantly lower in Amia compared with trout.  Opercular pressure amplitude, arterial blood pressure  and heart rate were not significantly different in the two species. differed between the two species. P  aQ2  .  Arterial blood 0  Blood gases  Amia had a significantly lower blood p H and  content was not significantly different (table 8).  2  These  differences are probably related to the difference in water temperature (10°C in trout vs 20°C in Amia), and possibly to the different ventilatory strategies and activity levels of the two species.  Amia had resting catecholamine levels in the  plasma about one order of magnitude higher than those in trout. NaCl infusion resulted in a small but significant increase in P , but no op  significant changes in f , P g  D A  or f  h  (fig 20).  Air-breathing frequency did not  change, remaining at 0.66 ±0.33 (mean ± S.E.) breaths/hr. At 5 minutes p.i., there was a significant reduction in C o , but no changes in p H or P o a  2  a  Circulating [NE] and [E] did not change significantly (table 9).  a  2  (table 8).  A representative  trace of the effects of NaCl infusion in Amia is shown in fig 15. NH4HCO3 infusion had no significant effects on P , f op  significantly increased P  and f .  D A  h  g  (fig 21) or f , but ab  At 5 minutes p.i., there was a small but  significant reduction in p H , but no changes in C o or P o (table 8). a  catecholamine levels did not change (table 9).  a  2  a  2  Circulating  121  Figure 20: Mean per cent change (± S.E.) in P , f , P DA  h  op  and f  following NaCl infusion in Amia. n = 5. C = control, shaded bar represents infusion period.  f  % change in fg —  I  Ol  o•  H  O o  1  % change in P p % change in f^ —  I Ol O o o  —  1  KJ  O o  — to  Ol oI o o Ol  ••••  —»  O o  h  tlllltllltttMIIIMtltl  •• *• •• •I  % change in P Q ^  0  • A*  I N>  —*  C n o o o Ol  H  h  H  h  MllMIMMMMIItMtll  • • • • • • • • • • • • • • • • • • • •  »V« • • • * • • • • • • • • • • • • • • • «  ° Ol  --  ( I  3 CD  CO  ro O "  to  O '  ro ro  123  Figure 21: Mean per cent change (± S.E.) in P , f , P DA  following NH4HCO3 infusion in Amia.  h  op  and f  n = 5  C = control, shaded bar represents infusion period.  $  % change in fg -* CTI o  H  % change in P p 0  ro o o  -*  ••• *  •t»•  I  T l-CH  •CM  i-CH  to On '  o '  —  n  KJ  h  ••tt•t•••••*i  O '  % change in f  • ••••••••••ft********** ~ »**«•••«••**«*•••••«•  % change in Ppj^  125  DISCUSSION The blood gas measurements in Series 1 and 2 allow analysis of the relative roles of pH , T a  a C 0 2  and T  a m m  in the ventilatory responses observed for trout.  The relationship between p H and ventilation: a  Following NaOH infusion there was a significant increase in blood pH, but this was not direcdy associated with any immediate increase in Vg.  This indicates  that increases in p H are not a direct ventilatory stimulant, and thus the increase in a  ventilation observed following N a H C 0  3  infusion (Janssen and Randall, 1975; fig  14) was not a result of the associated increase in p H . a  It is interesting that  infusions of NaOH at doses 1.5 or 2 times those reported here do not stimulate ventilation  in  a  pattern  similar  immediately lead to convulsions  to  that  following  NaHC0  3  infusion,  and death (unpublished observations).  but The  significant increase in ventilation seen 20 minutes following NaOH infusion is difficult to interpret, as no associated blood gas measurements were collected. Decreases  in p H resulting from infusion of HC1 or NH4CI are directly a  associated with ventilatory responses, a result similar to that observed by Aota et al (1990).  The relationship between T  a m m  , T  a C 0 2  and ventilation:  Infusions of NH C1 and NH^HCO;, stimulated ventilation significantly, and were 4  associated with increases in both T ^ and [NH ]. 3  The decrease in p H following a  NH^Cl infusion might have been responsible for the ventilatory increase seen, but  126 following N H H C 0 infusion, there was no change in p H . 4  3  a  Sodium bicarbonate infusion, however, also stimulated ventilation but was not associated with any significant increase in plasma that the response  was  or [NH ].  This indicates  3  stimulated by the measured increase in T  a C 0 2  .  The  ventilatory response occurred in the absence of a significant increase in  P  a C 0  2»  indicating that it was a result of increases in plasma HC0 " concentration ([HC0 " 3  ]).  The ventilatory response  increases  in  to N H H C 0 4  may also have been a result of  3  [ H C 0 ] , in addition to the increase in T 3  responses to NH C1, N H H C 0 4  4  and N a H C 0  3  3  3  a m m  .  The ventilatory  were not a result of changes in  plasma osmolality following infusion, because infusion of NaCl did not have any effects on ventilation.  These results indicate that water-breathing fish show a ventilatory response to increases in T the  cause  Ventilatory  a C 0 2  of  and T the  a m m  .  The evidence indicates that increases in [ H C 0 ] were 3  ventilatory  responses  to  NH^Cl  response  seen  and N H H C 0 4  following 3  NaHC0  infusions  are  3  infusion.  evidence  of  ventilatory sensitivity to ammonia, with a reduction in p H (NF^Cl infusion) or a  increases in [HC0 ~] ( N H H C 0 3  4  3  infusion) probably contributing to the response.  Possible mechanisms behind ventilatory responses: As stated in the Introduction, the primary source of ventilatory drive in waterbreathing fish is water and blood 0  Ventilatory responses to blood 0  2  status in fish appear to be more closely correlated with changes in blood 0  2  2  status.  content than with 0  partial pressure (Dejours, 1981; Smith and Jones,  2  1982;  Randall, 1982). It is conceivable that all the ventilatory responses observed in Series 1 and 2 were a result of a reduction in C that this was not the case.  a 0 2  .  The results of Series 3 indicate, however,  Following H Q infusion, C  declined significantly, as  a G 2  a result of the effects of a reduction in p H on blood 0 a  Bohr and Root effects.  carrying capacity, via  The observed ventilatory increase, therefore, was most  likely stimulated by the reduction in C Jones  2  a 0 2  ; results similar to those of Smith and  (1982) and Aota et al. (1990), for rainbow trout.  ventilatory responses to N a H C 0 a significant reduction in C  a 0 2  .  3  Thus, the effect of HC0 " and T ^ on ventilation 3  2  a significant reduction in P  observed ventilatory response. reduction in P o . a  blood 0  2  2  NH HC0 4  3  the  and NH4HCO3 infusion were not associated with  do not appear to be mediated by changes in C„o . there was  By contrast,  aG2  ,  Following N a H C 0  3  infusion,  which probably contributed to the  It is unknown why N a H C 0  3  infusion caused a  infusion, however, did not effect any changes in  status.  Cardiovascular responses observed in Series 3 also indicate that there is a difference in the characteristics of the response to HC1 and the responses to N a H C 0 and N H H C 0 . 3  4  3  HC1 has no effect on f , whereas N a H C 0 and N H H C 0 h  3  4  3  infusion both result in a tachycardia. In fish, catecholamines (NE and E) are released into circulation during stress (Nakano and Tomlinson, 1967; Perry et al., 1989).  Catecholamines  stimulate  ventilation in the eel, A. anguilla (Peyraud-Waitzennegger, 1979), and in trout  128 (Aota, pers. comm.).  There is evidence that the ventilatory responses to internal  acidosis are mediated by catecholamines in trout (Aota et al., 1990).  In the  present experiment, there was a significant increase in the levels of circulating N E and E associated with the ventilatory responses to HC1 and N a H C 0 . 3  This  suggests that the ventilatory responses to increases in [HC0 "] may have resulted 3  from release of catecholamines, which then stimulated peripheral or central sites involved in ventilatory control. Catecholamines are known to increase P  D A  and f in rainbow trout (Wood and h  Shelton, 1980), which might explain the cardiovascular responses observed in this study.  Increased acidity has a negative inotropic and chronotropic effect on the  isolated trout heart, that is offset by the effects of epinephrine (Farrell, MacLeod and Chancey, 1986).  Thus, following acid infusion, catecholamine release may  have ameliorated the negative chronotropic effects of an acidosis, resulting in no change in f .  In the absence of an acidosis, catecholamines release may have  h  stimulated heart rate, producing the tachycardia seen following N a H C 0 Following N H H C 0 4  3  3  infusion.  infusion, however, there was a tachycardia that was not  associated with a significant increase in [NE] and [E], It is unknown whether the ventilatory and cardiovascular responses to N a H C 0 actually represent direct evidence of sensitivity to [ H C 0 ] in trout. It is 3  3  possible that the infusions stimulated catecholamine release as a generalised stress response and the catecholamines then stimulated an increase in ventilation and cardiovascular variables  as  a secondary  effect.  The proximate reason for  catecholamine release following H C 0 ' loading is unknown, although release may 3  have been stimulated by the reduction in Po2-  In mammals, a reduction in Po2  a  a  will stimulate catecholamine release from the adrenal medulla (Nishijima, Breslow, Raff and Traystman, 1989). The ventilatory responses to NH C1 and NH4HCO3 occurred in the absence of a 4  catecholamine release or (in the case of NH4HCO3) of any change in blood 0 status, indicating that trout show a direct ventilatory response  2  to ammonia,  possibly similar to that described in mammals (Wischer and Kazemi 1974). It is conceivable that the increase in ventilation and heart rate seen following NaHC0  3  and N H H C 0 4  3  infusion functioned to aid in returning T  a C 0 2  or T ^ to  normal, by increasing passive diffusion of C 0 and N H from blood to water at 2  the gills.  3  Iwama, Boutilier, Heming and Randall (1987) showed that changes in  gill water flow had virtually no effect on P  aCQ2  , and the factor limiting C 0  2  excretion appears to be HC0 " dehydration by the red blood cell (Perry, Davie, 3  Daxboeck and Randall, 1982; Randall, 1990). not  determine  whether  C0  2  diffusion  might  significantly elevated above control values. immediately  following  NaHC0  3  However, Iwama et al. (1987) did limiting when  T  a C Q 2  was  At the dose used in this study,  and N H H C 0 4  be  3  infusion,  T  a C Q 2  would  approximately doubled and T ^ increased sevenfold, such that C 0  2  have  and N H  excretion may- have been transiently limited by gill water and blood flow.  3  It is  unknown whether trout would normally experience such large increases in T  a C 0 2  ,  so it is unknown whether observed ventilatory and cardiovascular responses are of any  functional significance.  There is evidence,  however, that plasma T ^  increases following feeding in sockeye salmon (Oncorhynchus nerka) (Brett and  Zala, 1975). In Amia, unlike trout, there was no ventilatory response to changes in H C 0 " or T 3  a m m  , but a ventilatory response to NaCl.  The ventilatory response to 2  M NaCl is presumably a result of the measured decrease in C  a 0 2  , and occurred in  the absence of a significant increase in [NE] and [E]. Amia did show an increase in P  D A  and f in response to NH4HCO3 infusion, similar to that seen in trout, which h  is presumably a response to increases in T  a m m  .  In conclusion, water-breathing fish (trout) show a ventilatory response increases in plasma T  a C 0 2  and T  cardiovascular response to T response to T demonstrate  a  a m m  a C O Z  a m m  .  There is evidence that the ventilatory and  is mediated by catecholamines, and the ventilatory  may be similar to that seen in mammals.  ventilatory  cardiovascular response to T  response a m m  to  to  either  T  a C 0 2  similar to that of trout.  or  T^,  Amia but  do not show  a  Chapter 4: The Effects of Branchial Denervation and Pseudobranch Ablation on Cardiovascular and Ventilatory Responses in Amia  132 INTRODUCTION Amia increases gill ventilation and air-breathing in response to aquatic hypoxia (Johanson, Hansen and Lenfant, 1970; Randall, et al. 1981), and as discussed in chapter 2,  air-breathing and gill  ventilatory responses to internal  disturbances appear to be more closely correlated with C  a 0 2  acid-base  than with pH . Thus, a  Amia is similar to water breathing fish, where the primary stimulus modality for ventilatory and cardiovascular reflexes is 0  2  (Randall and Jones, 1973; Dejours,  1973; Smith and Jones, 1982; Randall, 1982).  The putative sites of 0  2  sensitive  receptors responsible for hypoxic and hypoxaemic reflexes in Amia (and other airbreathing fish) remain controversial.  In teleosts, recent evidence suggests that  reflex responses to hypoxia are mediated by O -sensitive chemoreceptors in the z  gills and pseudobranch, innervated by cranial nerves VII, IX and X . of nerve activity sensitive to both internal and external 0  2  Recordings  levels have been  obtained from the vagus nerve to the first gill arch of tuna (Milsom and Brill, 1986) and the glossopharyngeal nerve to the first gill arch of trout (Burleson and Milsom, 1990).  Section of the IXth and Xth cranial nerves to the first gill arch  abolishes hypoxic bradycardia in salmonids (Smith and Jones, 1978; Smith and Davie, 1984) and cod (Fritsche and Nilsson, 1989). Various  studies employing  section  of  gill  nerves  failed,  however,  completely to abolish ventilatory responses to hypoxia in water breathing fish (Shelton, 1959; Hughes and Shelton, 1962; Saunders and Sutterlin, 1971; Butler, Taylor and Short, 1977) and in one air-breathing fish, the gar, Lepisosteus osseus (Smatresk, 1987). This may be because, as suggested by Smatresk (1990), in each  of these studies, the pseudobranch and/or nerves serving one particular gill arch were left intact.  Complete branchial denervation abolishes ventilatory responses to  hypoxia and NaCN in anaesthetized channel catfish. receptors appear to be located diffusely  In the catfish, 0  2  sensitive  throughout the gills, and even one  branchial nerve left intact unilaterally is sufficient  to stimulate a ventilatory  response (Burleson, 1986). Recent evidence suggests that circulating catecholamines (NE and E) might be responsible for mediating ventilatory responses to hypoxia and hypoxaemia in dogfish and trout, possibly via a direct effect on central respiratory motoneurons (Randall and Taylor, 1988; Taylor and Randall, 1989; Aota et al, 1990). There is evidence to suggest that catecholamines might also play a role in gill ventilatory responses to hypoxaemia in Amia (see chapter 2). This study employs total branchial denervation (branchial branches of IX and X) and pseudobranch ablation to assess the role of sensory input from the gills and pseudobranch in cardiovascular, air-breathing and ventilatory responses to hypoxia, NaCN, catecholamines  and acidaemia in Amia.  A conscious animal  preparation was used to avoid the effects of anaesthesia, which may compromise cardiovascular and ventilatory reflex responses.  134 MATERIALS AND METHODS  Amia weighing 400 to 1000 g were maintained and temperature acclimated as described in general material and methods.  Animal Preparation: Animals were anaesthetized in a tricainemethanesulphonate (MS-222) solution at a concentration of 1:10,000, buffered with sodium bicarbonate.  Following  transfer to an operating table, fish were artificially ventilated with a buffered M S 222 solution at 1:20,000, bubbled with 0  2  to help maintain P  aQ2  .  A dorsal aortic  (DA) cannula (PE 50, Intramedic) was implanted using the technique of Soivio, Westman and Nyholm (1972).  A buccal cannula was fitted using flared PE50  (Intramedic) passed through a small hole drilled in the roof of the mouth, and secured with a cuff and sutures.  A n opercular cannula was fitted, using flared  PE190 (Intramedic) passed through a small hole drilled in the operculum, secured with a cuff and sutures.  The operculum was reflected forward, and a small (1 to  1.5 cm) incision made in the epithelium dorsal and posterior to the fourth gill arch.  This allowed access to the branchial branches of cranial nerve X , serving  all four gill arches.  These were gently dissected free of connective tissue and  sectioned with iris scissors, care being taken not to damage gill vasculature, musculature or cardiac and visceral branches of X . The incision was closed with absorbable Vicryl coated polyglactin sutures (Ethicon 2.0).  Cranial nerve IX,  serving the first gill arch, was exposed by making a small (0.3 to 0.5 cm)  135 incision, at the base of the gill filaments, where the arch meets the roof of the opercular cavity, and dissecting the nerve free of connective tissue. was then sectioned with iris scissors.  The nerve  The pseudobranch in bowfin is glandular  and situated in the roof of the mouth just anterior to the first gill arch. exposed via a small (0.5 cm) incision and removed by cautery.  It was  The same  procedure was followed on the other side, so that denervation and pseudobranch ablation were bilateral. section  was  Surgery took between 30 and 40 minutes and nerve  confirmed post-mortem,  by dissection.  Sham-operated  (shams) had their gill nerves exposed as described, but not sectioned.  animals The fish  were transferred to individual, darkened plexiglass chambers (water volume 9 1), ventilated with water until ventilatory movements resumed and then allowed to recover for 48  hr with a continuous  mls/kg/min) through the chamber.  flow  of aerated water  (approx.  900  The anterior end of the chamber had an air-  space (volume 1.6 1), to allow air-breathing.  Protocol: Following recovery, V fish.  0 2  was measured in both denervated and sham-operated  Samples of inflow and outflow water were analysed for P , as described in G2  General materials and methods, and water V fish's weight and the water flow rate. decline in P  0 2  0 2  (V (w)) calculated, knowing the 02  To measure air-breathing V  0 2  (V (a)), the 02  in the anterior air-breathing chamber when sealed for a two to three  hour period was recorded, and V (a) calculated knowing the volume of the air02  space and the fish's weight.  Subsequently, the water-filled opercular cannula was attached to a pressure transducer  (Statham  P23BB), allowing  measurement  of  beats/min) and opercular pressure amplitude (P , cm H 0 ) . op  2  ventilation  rate  (f , g  The saline-filled D A  cannula was attached to a pressure transducer (Statham P23Db), for measurement of heart rate (f , beats/min) and D A blood pressure (P , cm H 0 ) . h  DA  2  The output  from both transducers was displayed on a pen recorder (Gould 8188-2202-XX). Air breathing frequency (f , breaths.hr) was visible as large pressure excursions on ab  the opercular trace, associated with changes in f and P . h  DA  Air breaths were all  verified visually through a small hole in a screen separating experimenter and fish. When required, 700 ul blood samples were withdrawn anaerobically from the D A cannula, via a three-way stopcock at the pressure transducer. Blood samples were replaced with an equal volume of heparinised saline.  500 u.1 of blood was  immediately centrifuged, the plasma decanted and frozen in liquid nitrogen for subsequent  analysis  of plasma catecholamine  materials and methods.  Blood p H (pH ), P a  0 2  levels, as described in general  (P ) and 0 a02  2  content (C ) were all a02  measured as described in general materials and methods. Ventilatory and cardiovascular responses to a variety of different stimuli were measured in shams and denervates.  Experiments on any one fish were performed  over a two day period, with overnight recovery between days.  No experiments  were initiated until ventilatory and cardiovascular parameters had remained stable for 30 minutes. randomly:  Animals were exposed to the following treatments,  assigned  137 Treatment 1: Using a three way valve at the inflow of the plexiglass chamber, animals were exposed to hypoxic water ( P bubbling N  2  w02  = 47.3 ± 1 . 2 mmHg) obtained by  counter-current to water flowing through a stripping column.  were exposed for 15 minutes, and then normoxic flow was resumed. samples  were  collected  immediately  before,  and after  5  minutes  Fish Blood  exposure.  Animals were then allowed a minimum of two hours recovery.  Treatment 2:  300 u.1 heparinised saline control and 300jig N a C N (dissolved in  300 ul saline) delivered as a bolus injection via the D A cannula.  Treatment 3:  1 ml saline control and lmg NaCN (in 1 ml saline) given as a  bolus injection into the buccal cavity via the buccal cannula.  Treatment 4:  0.5 ml/kg saline control, 0.5 ml/kg 10" M norepinephrine 5  hydrochloride (Sigma), in saline, and 0.5 ml/kg 10" M epinephrine bitartrate 5  (Sigma), in saline, infused via the D A cannula.  In  all treatments,  control and experimental  injections  or infusions  were  performed in random order. Animals were given a minimum of 30 min to recover between injections in treatments 2, 3 and 4. Upon completion of this protocol, denervates were given a 2.5 ml/kg control, saline infusion followed by 1 hr recovery and a 2.5 ml/kg 0.1 M hydrochloric acid (HC1) infusion, in a saline vehicle.  Infusions were performed as described in  138 general materials and methods.  Blood samples were withdrawn immediately  before and 5 min after each infusion.  Data analysis and statistics: Ventilatory and cardiovascular responses were analysed for a control period and at designated intervals following each experimental intervention.  Ventilation  and heart rate were counted for 30 seconds in each minute, and P  op  and P  D A  averaged from six measurements within that period; for two minutes control and at  1,  2.5,  5,  measurements  10  and  15  min following  were also taken at 30 min.  intervention.  For acid infusion,  Mean air-breathing frequency was  calculated for the 15 min period following intervention.  When a blood sample  was withdrawn, cardiovascular measurements were made at 4 min post-infusion. Cardiovascular and gill ventilatory responses were normalised as per cent change and, following arcsine transformation, compared at each time interval with an A N O V A , and compared "a posteriori" with the averaged control value.  In some  cases, gill ventilatory variables were compared between control and peak response using a paired t-test on normalised, transformed values. were used for graphical display.  Normalised responses  Control and experimental blood measurements  within a treatment were compared with a paired t-test.  Air-breathing frequency  was compared betwen control and experimental injections  or infusions  paired t-tests, and between shams and deneryates using unpaired t-tests.  using  139RESULTS. At 20°C, in aquatic normoxia, sham-treated Amia obtained 0.1 per cent of their total 0 0  2  2  uptake by air-breathing. Denervated animals had a significantly reduced  consumption rate (30 % lower than shams), and there was no 0  breathing (table 10).  Mean control values for P , f , P  2  uptake by air-  op  and f are in table 10.  There was no significant difference between denervates  and shams for these  DA  variables. During normoxia, f  h  g  was very low in the shams, usually zero, and only  ab  one denervate airbreathed, on two occasions (table 11). showed no differences in p H and C a  a Q 2  In normoxia, denervates  as compared with shams, but P  aQ2  was  significantly lower (table 12).  Effects of aquatic hypoxia: In shams, aquatic hypoxia elicited significant cardiovascular and ventilatory responses (fig 22).  A gradually developing bradycardia was evident, with f  h  significantly reduced following 15 min exposure. significant changes in P of hypoxic exposure.  op  Gill ventilation increased, with  and f at five minutes that were sustained until the end g  Air-breathing frequency increased significantly (table 11),  with most of the airbreaths occurring in the first five exposure.  Arterial blood p H and C  a 0 2  minutes  of hypoxic  were maintained during hypoxia, with no  change from pre-exposure values at five minutes post-exposure, but P  a 0 2  significantly (table 12). In denervates, the response to aquatic hypoxia was different (fig 22).  decreased  140 Table 10: Normoxic V  G 2  , pH , f , P a  h  op  and f  Shams  Denervates  v (t)  52.8±4.9  36.7±3.3 *  V (a)  0.06±0.02  0*  V (w)  52.7+4.9  36.6±3.3 *  PDA  28.8±0.8  29.8±0.5  f  30.0+0.8  25.6+0.3  0.66±0.05  1.56+0.08  12.2±0.6  10.7±0.4  02  02  02  h  P x  op  s  Values are means + S.E., * = significantly different (P=0.05) N = 6 for sham V ; N = 7 for denervate V Cardiovascular and ventilatory variables = mean of 48 measurements on 6 shams and 64 measurements on 7 denervates. Units: mgOi/kg/hr for V ; cm H 0 for P and P , beats/min for f and f . Q 2  0 2  0 2  2  D A  op  h  g  141 Table 1 1 : Airbreathing frequency (breaths/hr).  Shams  Denervates  Partial Denervates  Hypoxia  5.1+1.5  0 +  External saline  0  0.7+0.7  0  External NaCN  1.33±0.9  0.7±0.7  4.0+.1.3 *  Internal saline  0.7±0.7  0  Internal NaCN  0  0  NE infusion  0  0  E infusion  0  0  Saline infusion  -  0  Add infusion  -  0  Values = mean ± S.E. + = significantly different from sham hypoxia (P=0.05); * = significantly different (P=0.05) from saline injection. N = 6 for shams, 7 for denervates, 7 for partial denervates.  142  Table 1 2 : Arterial blood gases:  pH  a  Pa02  C 02 a  Sham Normoxia  7.72 +0.02  46.7 +12.0  4.45 ±0.76  Sham Hypoxia  7.73 +0.03  27.8* ±4.8  3.07 ±0.35  Denervate Normoxia  7.75 ±0.02  16.5+ ±1.6  3.80 ±0.68  Denervate Hypoxia  7.72 ±0.02  11.3* ±1.6  1.74* ±0.34  Denervate Pre-Saline Infusion  7.69 ±0.03  16.5 ±1.2  3.24 ±0.54  Denervate Post-Saline Infusion  7.68 ±0.03  17.2 ±1.3  3.48 ±0.60  Denervate Pre-Acid Infusion  7.68 ±0.02  17.0 ±1.8  3.25 ±0.64  Denervate Post-Acid Infusion  7.38* ±0.06  35.3* ±7.5  2.27* ±0.63  Values = mean ± S.E. * = significandy different from normoxic or pre-infusion; + = significantly different from sham normoxic. N = 6 for both shams and denervates Units: P = mmHg; C = vol.%. aQ2  a 0 2  Figure 22: The effects of aquatic hypoxia exposure on P , f , P DA  h  op  and f in sham-operated and g  denervated Amia. n = 6  The  bradycardia  response  was  abolished.  Gill  ventilation  attenuated, with no increase until ten minutes of exposure. sustained increase in f and a transient increase in P g  at 15 minutes.  There was no change in f  were abolished (table 11). pre-exposure values, but C  ab  op  responses  were  There was then a  that was no longer evident  during hypoxia, air-breathing responses  At five minutes exposure, p H was not changed from a  a 0 2  and P  a 0 2  were significantly reduced. In animals with  cranial nerve IX to the pseudobranch sectioned, air-breathing responses to hypoxia still occurred, and pseudobranch ablation was required to abolish the air-breathing response.  Effects of N a C N : Representative traces of the ventilatory and cardiovascular responses to N a C N in shams and denervates are presented in figure 23.  In shams, bolus injection of  N a C N into the buccal cavity (fig 24) elicited a transient bradycardia and a transient increase in P control.  op  at that time.  P  and f did not change significantly from  D A  g  In two out of six fish, external N a C N immediately stimulated an  airbreath (table 11).  It is of interest to note that partial denervates (i.e. animals  with a branchial branch of IX or X and/or the pseudobranch intact) showed an immediate and significant increase in f  ab  following external N a C N (table  11).  External saline control injections had no effect on any variable (fig 24). In denervates  (fig  25),  all cardiovascular and ventilatory responses were  abolished, with no change in any variable over time. One animal took an  Figure 23: Representative traces of cardiovascular and gill ventilatory responses to internal and external N a C N in shams and denervates.  147 External NaCN Sham 50 DA  25  cmH.O  T  P  cmH,0  o f — iwi^uvuvvn-  I . *— 2  _ , T NaCN  Denervate 50  25  cmH 0 2  o  P  " of  nH 0 cmH^  1^  2  T NaCN 1 min Internal NaCN Sham  p,  *  cmH  0  2p— o|— wwwvwv—vvWwwm^^ -2I—  t NaCN Denervate  P, cmH -2I—  t NaCN  148  Figure 24: The effects of externally applied N a C N on P , f , P DA  h  op  and f in sham operated animals, n = 6. g  150  Figure 25: The effects of externally applied NaCN on P , f , P DA  h  and f in denervated animals, n = 7 g  % C h a n g e in f  % C h a n g e in P  KM  H  •i  -- Q  OH  op  152 airbreath in response to both external saline and NaCN, in the latter case, at 13 minutes post-injection (table 11). on any variable (fig 25).  Saline bolus into the buccal cavity had no effect  Figures 24 and 25 are drawn to the same scale, to allow  comparison of responses by shams and denervates. Bolus injection of NaCN into the D A of shams had no significant effect on P and f , but significantly stimulated P h  op  and f (fig 26). g  P  op  D A  increased transiently at  2.5 minutes post-injection, and f was elevated at 1 and 2.5 minutes.  Figure 26  shows some evidence  were  g  significant.  of  cardiovascular responses,  Internal NaCN had no effect on f  ab  although they  not  (table 11), and a saline bolus into  the D A had no effect on any variable (fig 26). In  denervates  (fig  27)  the ventilatory responses to internal N a C N  abolished, with no significant changes in P  op  or f .  were  Cardiovascular variables  g  showed a similar trend to those of shams, but the changes over time were not statistically significant. the denervates.  There was no air-breathing response to internal N a C N in  Saline bolus had no effect on any variable (fig 27).  Effects of Catecholamines: In shams, N E infusion (fig 28) significantly increased P was no significant effect on f , and no stimulation of f . g  ab  DA  , f and P . There h  op  Blood pressure showed  a transient increase at 2.5 minutes, and f was elevated at 2.5, 5 and 10 minutes h  following infusion. Opercular pressure amplitude was significantly elevated at 2.5 minutes post-infusion, and remained elevated throughout the remainder of the  Figure 26: The effects of N a C N given in the D A on P , f , P DA  h  op  and f in sham operated animals, n g  155  Figure 27: The effects of NaCN given in the D A on P , f , P DA  h  and f in denervated animals, n = 7 g  157 measurement period.  E infusion had similar effects to N E on cardiovascular  variables, significantly increasing P  D A  from 1 minute post-infusion until the end of  the measurement period, but only transiently stimulating f , at 2.5 minutes. There h  was no statistically significant stimulation of P  or f  OP  g  by E , as measured by  A N O V A . However, a paired t-test showed a significant increase in mean P at 2.5 minutes post-infusion.  There was no stimulation of f  saline infusion had no effect on  PDA.5 fh» Pop» fg  or f  ab  (table 11).  OP  and f  g  Control  ab  (fig 28). In denervated fish (fig 29), N E had effects on P  D A  and f very similar to those h  seen in shams, but there was no statistically significant effect on P measured by A N O V A . sham response.  E also stimulated P  D A  OP  or f , when g  and f in a manner similar to the h  There was no significant effect on P  following E infusion, but f  OP  g  increased at 2.5, 5 and 10 minutes post-infusion, when measured by A N O V A . Whilst N E and E showed no statistically significant P A N O V A , a comparison of denervate mean control P  OP  OP  response as measured by  and f with mean P g  OP  and f  g  at 2.5 minutes post-infusion, by paired t-test, shows a significant increase in both ventilatory parameters at 2.5 minutes, which is evidence significant response.  of a physiologicaly  Saline control infusion had no effect on any measured  variable (fig 29).  Effects of acid infusion in denervates: HC1 infusion (fig 30) had a marked effect on P  D A  , which rose immediately and  was still significantly elevated at 30 minutes post-infusion, but f did not change h  significantly.  Gill ventilatory variables showed a large increase; P  OP  was  Figure 28: The effects of N E and E infusion on P , f , P DA  h  op  and f in sham operated animals, n = 6 g  control, the shaded bar represents the infusion period.  159  5  Time (mins)  10  15  O — O saline  • — • noradrenaline A — A adrenaline  160  Figure 29: The effects of N E and E infusion on P , f , P DA  h  op  and f in denervated animals, n = 6 g  C = control, the shaded bar represents the infusion period.  161  105--  :•  5  Time (mins)  10  15  o — o saline •• noradrenaline •A adrenaline  162 significantly elevated between 1 and 10 minutes post-infusion, and f increased at g  2.5 minutes and remained so until 30 minutes post-infusion. stimulation of air-breathing (table 11). were significantly depressed, and P infusion values (table 12).  a 0 2  There was no  At five minutes post-infusion, p H and C a  a 0 2  significantly elevated, as compared with pre-  Control saline infusion (fig 30) had no effect on  cardiovascular or ventilatory variables, and p H , C a  change as compared with pre-infusion values.  a 0 2  and P  aQ2  (table 12) did not  Figure 30: The effects of acid and saline infusion on P , f , P DA  h  op  and f in denervated animals, n = 6 g  control, the shaded bar represents the infusion period.  165 DISCUSSION The present study indicates that, in Amia,  section of all branchial branches of  cranial nerves IX and X , and extirpation of the pseudobranch, has marked effects on ventilatory and cardiovascular reflex responses to aquatic hypoxia, NaCN, catecholamine and acid infusion.  Resting cardiovascular, ventilatory and blood-gas variables: Following gill nerve section, a reduction or abolition of afferent information about water and blood 0  2  levels, and abolition of all efferent vascular and postural  motor control of the gill arches, probably combined to result in the measured reduction in V (shams).  Q 2  seen in the denervates as compared with sham-operated fish  Denervation did not change any resting cardiovascular or gill ventilatory  variables significantly.  Sham-treated Amia  did not airbreathe as much during  aquatic normoxia as noted in chapter 1, or by Johansen et al. (1970).  Denervated  animals did not airbreathe at all, except for one individual that did so on two occasions within one hour.  It is unlikely that denervation would abolish all air-  breathing behaviours, as afferent activity from stretch receptors in the swimbladder (Milsom  and  Jones,  1985)  may  stimulate  air-breathing  under appropriate  circumstances (branches of cranial nerve X to the swimbladder were intact), as is seen in gar (Smatresk and Azizi, 1989).  The reduction in P  denervates, as compared with shams, indicates a possible  a 0 2  in normoxic  ventilation:perfusion  mismatch, for the reasons stated above, but the fact that denervates had similar C  a 0 2  values to shams indicates that they were not hypoxaemic, and a normal pH,,  argues against a significant degree of lactic acid accumulation or anaerobic  166 metabolism.  Effects of denervation on cardiovascular responses: Abolition of the f response to hypoxia following gill denervation indicates that h  reflex bradycardia in Amia is controlled by receptors in the gills, as is the case in teleosts, i.e. salmonids (Smith and Jones, 1978, Smith and Davie, 1984), cod (Fritsche and Nilsson, 1989) and channel catfish (Burleson and Smatresk, 1990), where bradycardia is abolished by section of cranial nerves IX and X to the gills. In elasmobranchs cranial nerves V and VII (innervating the bucco-pharynx) also require sectioning (Butler, Taylor and Short, 1977). In shams, stimulation of a bradycardia by external NaCN, but not by internal NaCN  supports  previous  studies  that  indicate  that  0  2  receptors  mediating  bradycardia are externally oriented (Saunders and Sutterlin, 1971; Smatresk et al., 1986; Burleson and Smatresk, 1990).  The bradycardia was more immediate than  that seen during hypoxia, because NaCN represents a transient, supra-maximal stimulus. These responses are similar to the responses of channel catfish to N a C N (Burleson and Smatresk, 1990), but are in contrast to the responses of gar, where heart rate is either not changed by external or internal N a C N (Smatresk, 1986), or internal NaCN produces a bradycardia (Smatresk, Burleson and Azizi, although dosages were much higher in this study.  1986),  Abolition of all responses to  NaCN following denervation confirms that the externally oriented 0  2  receptors are  in the gills, innervated by cranial nerves IX and X . In shams and denervates, effects of catecholamines on both P  D A  and f  h  are  167  probably a result of the direct effects of catecholamines on the myocardium and peripheral vasculature in fish (Wood and Shelton, 1980; Farrell, 1983; Farrell, MacLeod and Chancey, 1986). Large increases  in P  and f  D A  h  following acid infusion in denervated fish  indicates that these responses are not mediated by 0  2  chemoreceptors in the gills.  The response is unlikely to be the result of catechoalmine release, because in intact Amia increases in P  D A  occur in response to acid infusion during hyperoxia  (see chapter 2), when there is no catecholamine release, indicating the possiblity of a direct p H effect.  It is possible that the increase in P  D A  is a result of  thromboxane or prostaglandin mediated effects, as acid infusion stimulates their release from erythrocytes in the cat, leading to increases in blood pressure (Shams, Peskar and Scheid, 1988)  Effects of denervation on ventilatory responses: Increases in P , f and f op  g  ab  following hypoxic exposure in shams are similar to  the response of most air-breathing fish (Smatresk, 1988). The shams did not show the hypoxic depression of gill ventilation noted by Johansen et al, Amia, and by Smatresk and Cameron (1982), in gar.  (1970), in  The lack of any air-  breathing by denervates during hypoxic exposure is evidence that 0  2  receptors  stimulating this behaviour are located in the gills or pseudobranch. This is similar to the lungfish, Protopterus aethiopicus (Lahiri, Szidon and Fishman, 1970) and to gar (Smatresk, 1987), where partial gill denervation attenuates the air-breathing response to hypoxia.  The fact that the air-breathing responses were abolished by  complete branchial denervation and pseudobranch ablation, but not by branchial  168 denervation and section of cranial nerve IX to the pseudobranch, indicates that cranial nerve VII to the pseudobranch must carry information adequate  to  stimulate air-breathing. The attenuated gill ventilatory response to hypoxia seen in this study, following complete denervation, may indicate the presence  of an  extrabranchial receptor (Bamford, 1974; Jones and Milsom, 1982).  It is also  possible,  circulating  however,  that  the  response  is  mediated  centrally  catecholamines, as suggested by Aota et al. (1990), for trout. stimulate gill ventilation in Amia  by  Catecholamines  but have no effect on air-breathing (see chapter  2), which would explain the absence of an air-breathing response in hypoxic denervates. In shams, N a C N given into the buccal cavity increased P , but had no op  significant effect on f or f . g  ab  This response is unlike that of gar (Smatresk, 1986),  where external N a C N stimulated air-breathing but had no significant effect on gill ventilation.  The lack of a significant air-breathing response in Amia  considered evidence that air-breathing is not controlled by 0 except that denervation abolishes the response. information from both internally and externally integrated to produce a final air-breathing pattern. in gar (Smatresk et al,  2  might be  chemo-receptors,  It is possible, however, that oriented receptor groups  is  This is known to be the case  1986), where internal receptors set the level of hypoxic  drive, and external receptors set the balance of air-breathing vs gill ventilation. In this  study,  during hypoxia, both  internal  and external  chemoreceptors  are  stimulated, leading to air-breathing.  External N a C N only stimulates externally  oriented  sometimes  receptors,  and  thus  only  stimulates  air-breathing.  In  169 incomplete denervates (i.e. animals with a branchial nerve and/or pseudobranch intact) external N a C N consistently stimulated air-breathing. may affect  Partial denervation  the balance of information from both groups, and lead to more  frequent airbreaths. Injections  of NaCN into the D A of shams elicited a similar ventilatory  response to that seen in gar (Smatresk, 1986), where internal N a C N does not stimulate air-breathing, but only gill ventilation.  This is unlike the response of  lungfish, where internally administered NaCN stimulates air-breathing (Lahiri et al., 1970). It is unknown whether internal and external NaCN injections stimulate the same or different groups of receptors in Amia, although two groups, oriented internally and externally, exist in gar (Smatresk et al., 1986). Complete denervation abolished all ventilatory responses to external and internal NaCN clearly indicating that the receptors responsible for mediating these reflexes are situated in the gills and pseudobranch, innervated by cranial nerves VII, IX and X . The abolition of all ventilatory responses to external and internal NaCN indicates that the P mediated by an 0  2  op  and f responses seen in hypoxic denervates are not g  chemoreceptor on the gills, and, indeed, that following gil  denervation and pseudobranch ablation, Amia does not exhibit any 0  sensitivity.  2  Infusion of N E into shams produced ventilatory effects similar to those seen in chapter 2 for intact fish, with increases in P  op  and f , but no change in f . g  ab  Following denervation, the ventilatory response to N E was no longer statistically significant, as measured by A N O V A , indicating that the P NE  op  and f responses to g  seen in Amia, and the ventilatory responses seen in the eel (Peyraud-  170 Waitzenegger, 1979) may be mediated to a large extent by receptors in the gills. The stimulation of f by E in denervates but not in shams is difficult to explain, g  but clearly indicates that E stimulates ventilation via an extra-branchial pathway. It should be noted that there is evidence of a P  response to N E and E in  op  denervates, which may be physiologically significant, if not statistically so. minutes post-infusion of N E and E , mean denervate P  op  At 2.5  and f were significantly g  higher than control, pre-infusion mean values (this was not the case for external and internal NaCN).  The absolute magnitudes of mean P  also similar to those seen in hypoxic denervates.  op  and f responses are g  Thus, a possible role for  catecholamines in mediating hypoxic gill ventilatory responses via a direct central effect, as postulated for trout (Aota et al., 1990) and dogfish (Taylor and Randall, 1989) may also occur in Amia. In denervates, the pattern of gill ventilatory responses and changes in blood gases following acid infusion was identical to that seen in intact animals (chapter 2), except that intact fish also showed a significant increase in f^. responses were mediated by a receptor sensitive to C  a 0 2  If the  (as suggested in chapter  2), then it is unusual that the receptor did not respond to NaCN, or stimulate airbreathing.  It seems unlikely that the P  op  and f  g  responses are mediated by  catecholamines, because the magnitude of the response to acid infusion was much greater than that seen following infusion of pharmacological levels of N E and E in denervates, unless there is a synergistic action between acid and catecholamines. It is possible that the responses are mediated by a p H receptor similar to that of air breathing vertebrates.  In that case, input from such a receptor must be  integrated with (and subordinate to) information from 0  2  receptors, because the  response to acid infusion in intact animals was abolished by aquatic hyperoxia (chapter 2).  Furthermore, the hypothetical receptor must only stimulate  gill  ventilation, as the air-breathing response to acid infusion was abolished in the denervates.  172  Summary: Denervation  of  the  gills  and extirpation  of  the  pseudobranch  abolishes  cardiovascular responses to hypoxia and NaCN, but not to catecholamine or acid infusion.  A l l air-breathing responses to experimental intervention were abolished  by denervation, as were gill ventilatory responses to NaCN.  Denervates exhibited  attenuated gill ventilatory responses to hypoxia and catecholamines, and a gill ventilatory response  to acid infusion similar to that of intact animals.  ventilatory responses seen in denervated  animals  may be  mediated  The by an  extrabranchial 0 receptor not sensitive to NaCN, by circulating catecholamines, or 2  by a p H sensitive receptor.  173 G E N E R A L DISCUSSION  This thesis has examined various aspects of the respiratory physiology of Amia. The overall results demonstrate that respiratory control in Amia is  essentially  similar to that of water-breathing fish, with the added capacity to breathe air. The results of the air-exposure experiment clearly indicate that Amia does not have the physiological capacities necessary to aestivate, being unable to detoxify ammonia as urea and reduce metabolism.  The swimbladder functions only to  sustain aerobic metabolism under purely aquatic conditions.  Amia has an unusual  gill structure (Daxboeck, Barnard and Randall, 1981; Olson, 1981), whereby the secondary  lamellae  arrangement.  of  adjacent  gill  filaments  are fused,  to form  It has been suggested (Daxboeck et al., 1981)  adaptation to survive air-exposure.  a lattice  that this is an  Blood perfusing the A B O in Amia first  traverses the gills and during air-exposure, collapse of the gills would occlude blood flow to the organ (and all systemic vascular beds).  This collapse can be  prevented by the lattice arrangement, and this might also allow the gills some role in gas-exchange in air.  The lack of any reduction in V  0 2  following air-exposure  does suggest that the A B O was still receiving an adequate blood supply, but clearly the unusual gill structure alone is not adequate to sustain long-term airexposure.  The inability of Amia to survive even moderate hypoxia without access  to air suggests that the existence of the A B O allows the fish to avoid reductions in aerobic metabolism, such that there was no selection pressure towards survival by anaerobic mechanisms.  174  The ventilatory sensitivity to reductions in C to that demonstrated in water-breathing fish.  a 0 2  demonstrated in Amia is similar  This C  a 0 2  sensitivity suggests that  Amia has internally oriented oxygen chemoreceptors functionally similar to those in the aortic body of mammals.  Mammals have two discrete groups of arterial  chemoreceptors, the carotid body, and a diffuse area of chemosensitivity in the aortic arch, the "aortic body" (Eyzaguirre, Fitzgerald, Lahiri and Zapata, 1986). All arterial chemoreceptors are sensitive to the P  0 2  at the receptor site.  carotid body has a very high, auto-regulated blood supply, such that the P receptor tissue is most closely correlated with plasma P  Q2  (P o2)a  does not have a high auto-regulated blood supply, and so the P tissue is affected by changes in blood 0 flow and C  a G 2  .  2  Q 2  The of the  The aortic body 0 2  at the receptor  delivery, which is the product of blood  Thus, aortic receptors respond vigorously to reduced blood flow,  anaemia and carboxyhaemoglobinaemia, whereas carotid receptors do not (Lahiri, 1980; Lahiri, Mulligan, Nishino, Mokashi and Davies, 1981). Indeed, as suggested by Smatresk (1990), it is only with the development of the highly specialised mammalian carotid body (or its avian analog) receptor sensitive primarily to P  a 0 2  that a  appears in vertebrate evolution, a receptor that  is functionally equivalent to the externally oriented chemoreceptors of fish. There is no evidence  for a structure similar to the carotid body in fish, and O  z  chemosensitivity appears to be diffusely organised throughout the gills and buccal cavity (Butler et al, 4).  1977; Burleson and Smatresk 1986; Smatresk, 1990, Chapter  It is possible that the periodic breathing behaviour of bimodally breathing  fish, and various other ectothermic vertebrate groups (Shelton et al, because these animals monitor blood oxygen delivery. apparent lack of a direct correlation between P  or P  a 0 2  1986) occurs  This would explain the a C 0 2  and breathing in these  animals (Shelton et al, 1986). Amphibian, reptile, avian and mammalian peripheral chemoreceptors all show sensitivity to P  a C 0 2  and p H (Ishii and Ishii, 1985 a,b; Piiper and Scheid, 1986; a  Eyzaguirre, Fitzgerald, Lahiri and Zapata, 1986).  In mammals, the response is  blunted but not abolished by hyperoxia (Eyzaguirre et al, 1986). The abolition of all ventilatory responses by hyperoxia in Amia different peripheral chemoreceptors, or that the P  suggests that these fish have a C 0 2  and p H response is entirely a  abolished by hyperoxia.  Catecholamine infusion stimulates ventilation in both water-breathers and airbreathers (Peyraud-Waitzennegger, 1979; Dempsey et al, Amia  exogenous catecholamines  stimulate  1986).  The fact that in  only gill ventilation  and not air-  breathing suggests that there are differences in the characteristics of air-breathing in Amia,  as compared with terrestrial air-breathers.  In mammals, catecholamines  stimulate ventilation largely by stimulating peripheral chemoreceptors (Dempsey et al,  1986; Eyzaguirre et al,  1986).  If this is the case in Amia  also, then the  receptors controlling gill ventilation may be similar pharmacologically to the peripheral chemoreceptors breathing  are  of terrestrial vertebrates,  probably separate  and those controlling air-  and pharmacologically  interesting, and warrants further study.  different.  This  is  176 There is evidence that the effects of catecholamines are exerted centrally. In mammals, catecholamines are largely inhibitory when applied centrally, and do not cross the blood:brain barrier in vivo (Dempsey et al., 1986).  In fish, however,  catecholamines cross the blood:brain barrier (Nekvasil and Olson, 1986), and when applied to respiratory neurones in the medulla stimulate ventilation (Taylor and Randall, 1990).  If the gill ventilatory response to catecholamines in Amia is  centrally evoked, then it suggests that air-breathing responses may be controlled by a different area of the brain. The fact that catecholamines do not stimulate airbreathing under moderate hypoxia, when there is a change in ventilatory pattern with an increased emphasis on air-breathing, indicates that the effects are exerted in an area not responsible for controlling air-breathing.  The  endogenous  release  of  catecholamines  seen following catecholamine  infusion in Amia also occurs in the American eel, Anguilla rostrata (Epple and Nibbio, 1985).  Catecholamines are released from chromaffin tissue, which in  actinopterygian fish is located in the posterior cardinal veins, just upstream of the heart (Nilsson, 1983). by  the  However,  Catecholamine release in fish usually requires stimulation  autonomic nerve  supply to  the  chromaffin  catecholamine-stimulated endogenous  tissue  catecholamine  (Nilsson,  1984).  release  in the  American eel does not require the presence of a brain or spinal cord (Hathaway, Brinn and Epple, 1989), suggesting that release can be effected by humoral influences in fish.  In mammals, stimulation of peripheral chemoreceptors effects a  reflex release of catecholamines from the adrenal medulla (Ungar and Phillips,  1983), but catecholamine release also occurs in the isolated adrenal gland in response to hypoxaemia (Nishijima, Breslow, Raff and Traystman, 1989).  This  suggests that the catecholamine release elicited by hypoxaemia in water-breathing fish (Perry et ah, 1989) and Amia may not require nervous stimulation.  If  catecholamine release in response to hypoxaemia in Amia is not a nervous reflex dependent on afferent information from chemoreceptors, but mediated humorally, then such a release may subsequently stimulate ventilation by a central effect, as postulated by Aota et al. (1990).  It is unusual that Amia do not show the ventilatory response to increases in T o2 or T , ^ seen in trout.  This may be because the dose used was not high  aC  enough, or because Amia are not as prone to exhibiting stress responses as trout. The  ventilatory responses by trout to N a H C 0  significant change in C  a 0 2  3  occurred in the absence of any  , but was associated with an increase in the level of  circulating catecholamines.  The stimulus for catecholamine release could be a  neurally mediated "stress" response, or possibly a direct effect of HC0 " on 3  chromaffin tissue.  The results do indicate, however, that in water-breathing fish  catecholamines may be able to stimulate ventilation in the absence of a reduction in C  a 0 2  .  It is interesting that N a H C 0 caused a significantly higher release of N E 3  than did HC1 infusion. The ventilatory response to ammonia in mammals may be an  evolutionary remnant of a response  ammonia in piscine ancestors.  to remove excesses of  endogenous  178 During aquatic hypoxia, Amia did not show the inhibition of gill ventilation or magnitude of air-breathing increase reported by Johansen et al. (1970) and reported by Smatresk et al. (1986) for gar. Amia does not show the vigorous airbreathing response to externally applied N a C N seen in gar (Smatresk, 1986), and require much higher doses of external and internal NaCN to elicit a ventilatory response.  This indicates that Amia are less sensitive to water and blood 0  2  status,  and may mean that Amia have different control mechanisms for air-breathing than those postulated for gar (Smatresk et ah, 1986).  Gar have reduced gills compared  with water-breathing fish (Smatresk and Cameron, 1982a) whereas Amia have gills similar in size to water-breathing fish (Daxboeck et al, 1981), which suggests that Amia do not rely on air-breathing to the same extent as gar. The evolution of air-breathing presumably required extensive re-organisation of the nervous system of fish.  It has been suggested that lungfish have two separate  central rhythm generators, one for gill ventilation and the other for air-breathing (Fishman, Galante and Pack, 1989). actinopterygian  fish  is  a  Other authors suggest that air-breathing in  re-organisation of coughing and suction-feeding  movements; requires relatively little neural re-organisation (Liem, 1980; Smatresk, 1990), and is critically dependent on afferent feedback (Shelton et al, Smatresk, 1990).  1986;  In Amia, following gill deafferentation, air-breathing did not  occur (except in one case) in normoxia, and responses to hypoxia, N a C N and hypoxaemia were abolished, indicating that air-breathing rhythmicity and responses are dependent to a large extent on afferent information from the gills, and probably also the swimbladder.  In Amia, the pseudobranch contains receptors that elicit air-breathing responses, with the information carried in cranial nerve VII, because section of cranial nerve IX to the pseudobranch was not sufficient to abolish air-breathing responses to hypoxia or external NaCN, but extirpation abolished the responses.  It would be  interesting to determine whether pseudobranch ablation alone would abolish all air-breathing responses, since the pseudobranch in Amia is unusual and glandular, with different vascular relationships than that of teleosts (Allis, 1897). Hedrick and Jones (1990) report that Amia show two different air-breath types, and suggest that one is driven by stretch-receptor input, and the other by a chemoreflex.  This  suggests that the individual Amia  that air-breathed following  branchial denervation may have done so in response to stretch-receptor input, as the responses were not associated with experimental intervention. The significant attenuation of gill ventilatory responses to  catecholamines  following gill denervation suggests that, similar to the case in mammals (Dempsey et al,  1986;  Eyzaguirre et al.,  1986), much of the ventilatory response  catecholamines is mediated peripherally. evoked  centrally.  This evidence  to  The remaining response is presumably  further indicates  that peripheral receptors  stimulating gill ventilation and air-breathing are pharmacologically and spatially separate, and that the air-breathing response is not integrated at the level of the gill ventilatory rhythm generator. ventilatory  response  was  an  In denervated animals, the most pronounced increase  in  ventilation  frequency  following  epinephrine infusion, whereas catecholamines exert their major effect on opercular pressure in intact animals.  The ventilatory response to hypoxia in denervates was  180 largely a result of an increase in opercular pressure, suggesting that there may be differences in the responses to epinephrine and hypoxia in the absence of afferent information from the gills. pronounced effect  It is difficult to explain why epinephrine had a more  on ventilation following  denervation, since norepinephrine  crosses the blood:brain barrier more freely in water-breathing fish (Nekvasil and Olson, 1986). The  vigorous  gill  ventilatory  response  to  acid infusion  following  gill  denervation suggests that Amia may have a central pH-sensitive receptor similar to that of mammals, but subordinate to oxygen reflex responses.  Indeed, the  attenuated  a result of  gill  ventilatory response  to hypoxia may have been  stimulation of this receptor, since the onset of the ventilatory response occurred five minutes following blood sample collection.  Interestingly, perfusion of the  medullary region of the cranial space in Amia with mock cerebrospinal fluid at various pH levels does not stimulate gill ventilation (Hedrick, Burleson, Jones and Milsom, unpublished data).  This indicates that the p H response seen in the  denervates may not be central in origin, or that the mock C S F used by Hedrick et al. did not communicate with the sites responsible for the p H response. a  In conclusion, Amia calva appears to be an aquatic animal with no ability to survive  prolonged  air-exposure.  The A B O functions  to  metabolism and blood oxygen delivery during aquatic hypoxia. not exhibit a ventilatory response to p H and P a  ventilatory sensitivity to increases in T  a C 0 2  and T  aC02  a m m  -  maintain  aerobic  Intact Amia do  Amia do not show the  seen in water-breathing fish.  181 Air-breathing and gill ventilation are stimulated by hypoxia and hypoxaemia, and receptors controlling the responses are in the gills.  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