RESPIRATORY AND CIRCULATORY CHANGES IN THE LOBSTER (HOMARUS VULGARIS) DURING LONG TERM EXPOSURE TO MODERATE HYPOXIA

J. exp. Biol, (1978), 73. 131-146 131 With 8 figures Printed in Great Britain RESPIRATORY AND CIRCULATORY CHANGES IN THE LOBSTER (HOMARUS VULGARIS) DURING LONG TERM EXPOSURE TO MODERATE HYPOXIA BY P. J. BUTLER, E. W. TAYLOR AND B. R. MCMAHON* Department of Zoology and Comparative Physiology, The University of Birmingham, Birmingham Bi$ 2TT, U.K. (Received 4 July 1977) SUMMARY 1. Within a few hours of the lobsters being placed in the experimental chamber in normoxic water (P Oi, 130-135 mmhg), their heart rate, oxygen uptake (^Oj) and ventilation volume (V u ) were relatively high. The scaphognathites were simultaneously active on both sides (bilateral ventilation) for 50% of the time, active on one side only (unilateral ventilation) for 32% of the time and simultaneously inactive (respiratory pause) for 18% of the time. Percentage extraction of O 2 (% Ext.) was relatively low. 48 h after having been set up, heart rate, V Oi and V g had decreased significantly, % Ext. had increased and bilateral ventilation occupied 25 % of the time. Heart rate was low and the beat irregular. 2. Upon subsequent exposure to hypoxia (P Oj, 50-55 mmhg), P Qi o, fell, there was initial hyperventilation, bilateral ventilation for 89 % of the time, heart beat was more regular but ' Oa did not change significantly. An initial increase in pha caused the O 2 affinity of the Hey to rise. After 72 h exposure to hypoxia j and pha had declined, but *V g was still higher than at the end of the period of normoxia. Blood lactate, P Oi and % Ext. were not significantly different from the values recorded after 48 h in normoxic water. Upon return to normoxic water, V g and the time the animals spent ventilating both branchial chambers fell, but P Ot increased. 3. It is concluded that for settled lobsters, I^Oa could be maintained during moderate hypoxia by increasing J, maintaining a regular heart beat, and at least initially by increasing the O 2 affinity of the Hey. INTRODUCTION The question as to whether a particular animal is a conformer or a regulator with respect to its oxygen uptake (^o 2 ) as environmental oxygen tension falls, has been the subject of numerous investigations (see Hill, 1976). During recent years, it has become clear that an individual species can be either a regulator or a conformer depending upon a number of factors. For example, at relatively high environmental temperatures an animal may be a conformer whereas at lower temperatures it may be a regulator (Spitzer, Marvin & Heath, 1969; Butler & Taylor, 1975). The experimental procedure also appears to be of paramount importance. Using fettered lobsters, Spoek (1974) # On study leave from Department of Biology, University of Calgary, Calgary, Alberta, Canada TzN 1N4.

132 P. J. BUTLER, E. W. TAYLOR AND B. R. MCMAHON was able to obtain almost identical results to those presented by Thomas (1954), whose animals were also restrained. In this case, Homarus vulgaris was classified as a conformer. However, at the same environmental temperature, Spoek found that free, inactive lobsters had a lower initial oxygen uptake and this remained virtually unchanged down to an environmental P Oa of approximately 30 mmhg. Under these conditions the animal was a regulator. Even in restrained animals, lownormoxic values of J^Oi and other variables can eventually be obtained if the animal is allowed sufficient time to settle in the experimental chamber, in which case subsequent exposure to mild, chronic hypoxia causes no reduction in V Oj (McMahon, Burggren & Wilkens 1974). In the present investigation, unrestrained lobsters {Homarus vulgaris) were allowed time to settle in the experimental apparatus before being exposed to hypoxia for several days. The object was to substantiate that this animal can maintain its oxygen uptake under such conditions and then to study the respiratory and circulatory changes that occur to make this possible. MATERIALS AND METHODS Lobsters (Homarus vulgaris) of either sex were used. The animals were in intermoult and they were fed regularly except during the period of the experiment. Twenty-one of the experiments were performed at the Gatty Marine Laboratories, St Andrews, and the remainder were performed on lobsters which were either obtained from St Andrews or from a commercial dealer and transferred to aquaria in Birmingham. In each case, the lobsters were acclimated to a temperature of 15 + 1 C (range) for at least 1 week before the experiments and all experiments were performed within this temperature range. Three different series of experiments were performed on 26 lobsters whose mass ranged from C22 to 0-51 kg, but the following schedule was similar for each series. The lobsters were prepared for the experiment, allowed 46-48 h to settle in normoxic sea water (P Oj, 130-135 mmhg) exposed to hypoxia(p Oa, 50-55 mmhg) for 9o-i2oh and then returned to normoxia for a further 48 h. In each series, great care was taken not to disturb the lobsters. The sea water was filtered and circulated from a large (1000 1) reservoir. The lowp Oj was obtained by bubbling nitrogen gas through the sea water in the reservoir. The P Oj was monitored and controlled by an E51443/O oxygen electrode, a TOX 40 oxygen transmitter and a CME 40 on/off controller (Radiometer Ltd). The P Oj range was set by the on/off controller which operated a solenoid valve situated in the nitrogen supply line. Thus the hypoxic P Oj was maintained within the range of 50-55 mmhg for the required period of time. In the first series of experiments, oxygen uptake was monitored from 12 lobsters and in four of these animals, inspired PO^PJ.O,,) an< i mixed expired Po,(Ps,o 8 ) were also measured so that ventilation volume (P g ) could be calculated using the Fick principle. The lobsters were placed in a cylindrical, continuous flow respirometer (cf. Butler & Taylor, 1975), which was surrounded by sea water at 15 C, and partially covered with a sheet of black polythene. Thus, the lobster was in dim light, experienced a natural photoperiod, yet could not see the experimenters. A constant rate of water flow (between 100 and 150 ml min -1 ) was maintained through the respirometer during an experiment and was measured using a glass flowmeter tube (Glass Precision Engineering Ltd, Hemel Hempstead). Water samples were withdrawn from

Long term hypoxia in the lobster 133 Cannula and tube for collecting mixed expired water Tip of cannula used to collect inspired water Fig. 1. Illustration of a lobster to show the positions of the cannula and tube used for collecting mixed expired water and of the cannula used for collecting inspired water. the inlet and outlet tubes of the respirometer and their P Oa was immediately measured by a Radiometer PHM 71 analyser and an oxygen electrode which was housed in a cuvette at 15 C (see Butler & Taylor, 1971). From these values of P Oj, the solubility coefficient for O 2 in sea water at 15 C (Harvey, 1955) and the flow rate of water through the respirometer, it was possible to calculate the oxygen uptake of the animal (I^oJ. All values of J^ have been corrected to S.T.P.D. Mixed expired water was collected by positioning an open-ended mask in front of both exhalant apertures (Fig. 1). The mask consisted of a 3 cm length of polyethylene tubing (i.d. 12 mm, wall thickness 2 mm) which was held in position by latex rubber that was shaped to pass between the second and third maxillipeds. Twoflapsof the rubber were fixed to the sides of the carapace with Eastman 910 cement (Ciba-Geigy). Thus, exhalant water from both branchial chambers passed through the polyethylene tube. This arrangement left the eyes, antennules, antennae and third maxillipeds completely free and did not obstruct or constrict the exhalant openings. Despite the fact that this arrangement obviously prevented feeding, a lobster at 10 C remained alive and, as far as is possible to tell, healthy for 3 months with this mask in place. A water sample was taken from this mask during the experiments via a length of cannula, one end of which lay in the centre of the polyethylene tube. This cannula passed over the rostrum between the eyes and was fixed to the dorsal surface of the carapace together with another length of cannula, one end of which lay at the base of the first pereiopod (Fig. 1). The cannulae were led to the outside of the respirometer so that samples of inspired and mixed expired water could be obtained without disturbing the animal. Water samples were taken at 1-3 h intervals throughout the day for each animal and mean values of F Oa and V a for each successive set of samples from the commencement of the experiment were computed. A sample of these repetitive means for Xf g is shown in

134 P. J. BUTLER, E. W. TAYLOR AND B. R. MCMAHON p,. 0!(mmHg ) 130-135 50-55 130-135 80 u 2 c g I Hea 60 40 20 0 1200 r - 1 1100 1000 = 900 In 800 700 volum ion Ventilat 600 500 400 300 200 100 24 48 72 96 120 144 168 192 Time(h) Fig. 2. Graphs to indicate the degree of variability in heart rate and ventilation volume in lobsters throughout the experimental period. Mean heart rate ( ) and its range (vertical lines) were obtained from one cj lobster of mass 024 kg. Mean values for ventilation volume (O) were obtained from four lobsters. The points joined together represent the values obtained during one calendar day at a particular P/, o a and contribute to the ' grand mean' values shown in Fig. 3. Fig. 2. One calendar day's worth of values at any onep />Oa were grouped together to produce the 'grand mean' values that are shown in Fig. 3. The time from the beginning of the experiment allocated to each of these grand mean values is the midpoint of the day's sampling times at a particular Pi,o*- In the second series of experiments, nine lobsters were used to record changes in heart rate and the frequency of scaphognathite beat. Heart rate was measured in eight animals as the E.C.G. Scaphognathite beat frequency was determined by measuring branchial chamber pressure via a fluid-filled cannula inserted into the branchial chamber which was connected to an Elcomatic EM 750 pressure transducer. The pressure in one branchial chamber was recorded in two lobsters, together with heart rate, whereas in one lobster, the pressures in both branchial chambers were measured simultaneously. The latter animal also had a mask attached to it so that measurements of PE,O, could be related to the various type of ventilatory activity that it displayed (see

Long term hypoxia in the lobster 135 later). Branchial chamber pressures and E.C.G. were recorded on an ink-writing pen recorder (George Washington Ltd). After preparation, each animal was placed into a tank of running sea water which contained a length of opaque plastic pipe into which the lobster crawled and then stayed for the majority of the experimental period. Measurements were made at various times throughout the experiment, and in particular just after the lobster had been prepared, just after exposure to hypoxia and just after return to normoxia. For the purpose of presenting this information, heart rate and the pattern of ventilation were determined at the times allotted to the 'grand mean' values for P Oi and J^. Eight measurements of heart rate were taken over a 30 min period and combined to give a mean value for each individual lobster. The mean and range for heart rate from a single animal can be seen in Fig. 2. The mean values from all of the lobsters were then combined to give a 'grand mean' value. As with the other series of experiments, great care was taken not to disturb the animals in any way, although on occasions it was necessary to replace broken E.C.G. wires. From the lobster in which pressures in both branchial chambers were measured, it was apparent that the left and right scaphognathites did not always beat simultaneously or continuously. Measurements were taken over 75-150 min periods of the percentage time that the scaphognathites were simultaneously active on both sides (bilateral ventilation), active on one side only (unilateral ventilation) and simultaneously inactive (respiratory pause). During the final series of experiments, post-branchial haemolymph samples were obtained from the pericardial cavity. A hole was drilled through the calcareous exoskeleton above the pericardial cavity, just posterior to the position of the heart. The hole did not penetrate the hypodermis and it was covered by a resealing rubber membrane which was held in place with Eastman 910 cement. The position of the hole in the exoskeleton was marked on the rubber membrane and a haemolymph sample was obtained by piercing the rubber with a hypodermic needle and carefully inserting the needle just into the pericardial cavity. The required volume of haemolymph was drawn into a cold, glass syringe. When performed with care, this procedure did not noticeably disturb the animal. Because the haemolymph clots so readily, it was not possible during the present long-term experiments to use the method described by Taylor, Butler & Sherlock (1973) for obtaining post-branchial blood, whereby a cannula was in contact with the pericardial cavity. Preliminary investigations showed that it was not possible to obtain prebranchial haemolymph without considerably disturbing the lobsters. Thus, no attempts were made to obtain venous haemolymph during any of the present experiments. The following measurements were made on 07 ml of post-branchial blood. Arterial P o> (P a< 0 J and ph (pha) were measured with a Radiometer acid/base analyser (PHM 71) and a blood microsystem (BMS 3 Mk II) which housed the appropriate electrodes and thermostatically controlled their temperature at 15 C. The oxygen electrode was calibrated with deoxygenated and aerated sea water and the ph electrode with precision buffers. Oxygen content of the haemolymph was measured by a Lex-O 2 -Con oxygen analyser (Lexington Instruments Ltd). The glassware of this machine was kept free of protein deposits by frequent cleaning and it was calibrated with aerated, distilled water. 40 /il of haemolymph were used for each measurement. Lactic acid concentration in the haemolymph was determined enzymically (see Sigma bulletin 826-U.V.), some

136 P. J. BUTLER, E. W. TAYLOR AND B. R. MCMAHON days after a 150/41 sample had been 'fixed' with 8% perchloric acid and refrigerated. The assays were performed on a Beckman model 25 spectrophotometer. From the first two series of experiments, it was clear that the lobsters had completely settled in the experimental apparatus after 48 h. The measurements on postbranchial haemolymph were made 48 h after a lobster had been prepared and placed in aerated, circulating sea water; at 2 h, 18 h, 66 h and 114 h after exposure to hypoxia and then at 2 h, 24 h and 48 h after its return to normoxia. In two, large lobsters (mass 3-0 and 3-3 kg), 5 ml of post-branchial haemolymph were withdrawn 48 h after the animals had been in normoxic sea water and then again after 3 h of hypoxia. An oxygen equilibrium curve was determined for each haemolymph sample at 15 C and at the prevailing ph of the arterial haemolymph- This ph was obtained by adjusting the P COa in the equilibrating gas mixture. For the normoxic sample P COa was 3 mmhg, whereas for the hypoxic sample it was 1-5 mmhg. The curves were constructed by equilibrating the haemolymph in an intermittently spinning tonometer with accurately known, humidified gas mixtures which had been produced by gas mixing pumps (Wosthoff, Bochum). The oxygen tension, ph and oxygen content of a sample of the haemolymph were then measured as described above, for each equilibration mixture. Means of the measured variables are often expressed + s.e. of mean. Students' Mest was used to test the significance of any difference between two mean values. The word 'significant' in the present report means significant at the 95% confidence level (P < 0-05). RESULTS Normoxia Within 2-4 h of the animals being placed into the experimental chamber, heart rate, V g and V o% were relatively high (Figs. 3, 4) and heart beat was regular (see Figs. 2, 5). Percentage extraction of oxygen from the water (% Ext.) was low (Fig. 3). The animal ventilated bilaterally for 50% of the time, unilaterally for 32% of the time and for the remaining 18% of the time no ventilatory activity took place at all. During bilateral ventilation at this time, scaphognathite frequency was 112 min" 1 on both sides. During the next 20-22 h, there was a steady decline in heart rate to an average of 41 % of its initial value. Heart beat also became irregular with pauses of up to 20 s duration (Figs. 2, 5). Ventilation volume fell to 47% of its initial level and % Ext. increased by 70%, whereas there was no significant change in V Oi. Within the following 24 h, there were no further significant changes in heart rate, V g and % Ext.; there was, however, a significant reduction in V Oi to 80% of its initial value. The activity of the scaphognathites also changed: 36 h after the animal had been set up, bilateral ventilation occurred 25 % of the time and the frequency of scaphognathite beat during this time had fallen to 72-92 min" 1. Unilateral ventilation, involving either the right or the left scaphognathite, occurred 57 % of the time at a frequency of 95-128 min" 1 and respiratory pauses occupied 18% of the time (Figs. 4, 6). The duration of these respiratory pauses ranged from 1-5 to 8-6 min. Following a pause in ventilatory activity, both scaphognathites became active at very high frequencies (112-150 min" 1 ) for a short period. From the animal that had both branchial chambers cannulated and a respiratory

Long term hypoxia in the lobster 130-135 50-55 130-135 72 96 Time (h) Fig. 3. Mean changes in % Ext., ventilation volume and oxygen uptake in lobsters allowed to settle in normoxic water for 48 h, followed by exposure to hypoxic water for 90 h and subsequent return to normoxia. Number of animals (n) contributing to each set of values is given at end of each line. Figures in parentheses indicate number of observations, contributing to each point and a number refers to each successive point with no number. Vertical lines associated with each point are +S.E. of mean. Where vertical lines are absent, the s.e. of mean is within the limits of the symbol. mask fitted, there was a clear relationship between activity of the scaphognathites and P Et Oa. For example, at a P T Ot of 140 mmhg, P E Oa varied between 74 and 86 mmhg during bilateral ventilation, whereas during unilateral ventilation at a Pj,o, of 150 mmhg, P E Oa was 115 mmhg. During a long respiratory pause, measured P Et Oa gradually increased from 112 mmhg, 2 min after the onset of the pause, to 138 mmhg 6 min into the pause as the surrounding water slowly penetrated into the tube of the respiratory mask. Thus, for the four animals in the respirometer tubes, there would have been times, particularly towards the end of the normoxic period when respiratory pauses may have occupied 20 % of the time, when the animals were not ventilating at all. Yet for the reason illustrated above, P EOa would not have been equal top 7 Oa, and because of the lag within the respirometer, calculated J^ was never zero. Under these circumstances, the calculated values of % Ext. (which is derived from P E<0^ and perhaps more so of l (which is derived from %Ext. and V o^) are spuriously high.

138 P. J. BUTLER, E. W. TAYLOR AND B. R. MCMAHON P, Oi(mmHg) 80 130-135 50-55 130-135 u 2 c 60 40 20 \ \. 0 100 50 24 48 72 96 Time (h) 120 144 168 Fig. 4. Changes in heart rate and ventilatory activity in lobsters allowed to settle in normoxic water for 48 h, followed by exposure to hypoxia for 90 h and subsequent return to normoxia. Mean heart rate ( ) obtained from 48 observations on six lobsters. Vertical lines associated with each point are + S.E. of mean. Where vertical lines are absent, the S.E. of the mean is within the limits of the symbol. Ventilatory activity from one 2 lobster of mass 032 kg is expressed as the percentage of total time that the animal ventilated bilaterally (Q), that it ventilated unilaterally (g>) or during which there was a respiratory pause, i.e. during which there was no ventilation at all ( ). (a) (*) to (d) 1 III nil MUUI Mr Illl 1111 UUUUI IIIIIHII III 111 Hi IfflWmm uuuuhui UUtUilt III Illlllllll HI in IIII 1 Illlllll UhhlL it) HtH Illdllll Fig. 5. Sections of original traces from two lobsters to illustrate the heart beat frequency; (a) 3 h after placing animals in normoxic water; (6) 44 h after placing animals in normoxic water; (c) 12 h after subsequent exposure to hypoxia; (d) 2 h after return to normoxia. In each series, traces are from above downwards, E.C.G. from J lobster of mass 0-32 kg, E.C.G. from <J lobster of mass 0-24 kg, time marker (is s).

Long term hypoxia in the lobster (a) rom (Is) (*) w. (15 s) (Is) (15 s) (15 s) (d) (e) 'ffrf vpw <Wmvvvvyv«y#vvrtwv^^ (Is) (15 s) Fig. 6. Original traces from $ lobster of mass 0-32 kg showing simultaneous recordings of pressure in both branchial chambers, (a) 3 h after placing the animal in normoxic seawater, it spent long periods ventilating both branchial chambers. Traces (6) and (c) show the more variable ventilatory activity in the animal after it had been in normoxic sea water for 40 h. At the beginning of (6) there is a respiratory pause which had lasted for 6 min, there is then a 2 min period of bilateral ventilation and at the end of (6) one scaphognathite ceases its activity and does not recommence until 28 min later (c). During subsequent hypoxia the animal ventilated almost continuously on both sides (d), whereas upon return to normoxia, the animal spent most of its time ventilating one branchial chamber only (e). In each series, traces are, from above downwards, pressure in left branchial chamber, pressure in right branchial chamber, time marker (1 s or 15 s as indicated). As the pressure traces were used only to indicate respiratory frequency they were not calibrated. Note the brief periods when the pressure becomes more positive which signify reversals of the scaphognathite beat (Wilkens & McMahon, 1972). Thus under normoxic conditions, quiescent, settled lobsters (48 h after being set up) typically had an irregular heart beat and predominantly unilateral scaphognathite activity. Mean values of the measured variables at this time are given in Table 1. From Fig. 8 it can be seen that the P 50 of haemolymph taken from a large settled lobster under normoxic conditions was 14 mmhg at a ph of 7-67. One lobster maintained a high heart rate of 29-38 beats min" 1 throughout the last 24 h of normoxia whereas during the same period another, more typical animal, showed a variation in heart rate between 8-36 (see Fig. 2). The haemolymph of the former animal had an oxygen content when equilibrated with air of 0-9 vol. %, compared with 1-45 vol. % in that of the latter. This observation agrees with the findings of Spoek (1974) relating heart rate to the concentration of Hey in lobsters.

140 P. J. BUTLER, E. W. TAYLOR AND B. R. MCMAHON 48hin normoxic water Hypoxia Normoxia -6 0 72 96 120 144 168 Time(h) Fig. 7. Mean values of P a, 0. 2, C a> o a» pha, blood lactate and P, t Oa from five lobsters after having settled in normoxic sea water for 48 h, at various times during subsequent exposure to hypoxia, and upon return to normoxia. Vertical lines associated with each point are ± s.e. of mean. Where vertical lines are absent, the s.e. of mean is within the limits of the symbol. Hypoxia The most noticeable, immediate responses upon exposure to hypoxic water (P 7 o 2, 50-55 mmhg) were a significant reduction in P a> Oa and significant increases in both V g and pha, with V g reaching 2-2 x its settled, normoxic value (Figs. 3, 7). Associated with the rise in pha, there was a reduction in P 50 of the haemocyanin. (Fig. 8). In the lobster that contributed to Fig. 8, P 50 fell to 8 mmhg. The rise in X? Q was related to a dramatic increase in the time that the animal spent ventilating both branchial chambers, together with a rise in scaphognathite frequency to 102-140 min" 1. There were virtually no respiratory pauses and unilateral ventilation occupied 10% of the time (Fig. 4). This situation continued for the first z\\i of hypoxia. There were no significant changes in % Ext., P o>, C aoa or haemolymph lactate concentration upon initial exposure to hypoxia; % Ext did not alter throughout the period of hypoxia whereas both C Oi Oa and blood lactate did fall significantly after 20 h (Figs. 3, 7). Heart rate showed a slight but significant reduction and heart beat became less irregular (Figs. 2, 4, 5). There was no further change in mean heart rate

Long term hypoxia in the lobster 141 1-6 1-4 1-2 10 1 0-8 0-6 0-4 0-2 0 10 20 30 40 50 60 70 P,, Ol (mmhg) Fig. 8. Oxygen equilibrium curves for whole blood of a cj lobster of mass 3-0 kg, 48 h after the animal had been allowed to settle in normoxic sea water (P/,o a, 130 mmhg) and 3 h after subsequent exposure to hypoxia CP/,o 2 55 mmhg). The curves were constructed at the prevailing ph of arterial blood which was 7-67 during normoxia (V) and 7-85 during hypoxia (#). ^co a fthe blood was 3 mmhg and 1-5 mmhg respectively. throughout the period of hypoxia, although the beat did become progressively more regular (Fig. 2). After 48 h of hypoxia, l showed a significant reduction, and this was a reflexion of a reduction in the time that the lobster spent bilaterally ventilating (Figs. 3, 4). Accompanying the fall in V g there was also a reduction in ph a so that after 72 h of hypoxia it was not significantly different from its settled, normoxic level. After approximately 120 h of hypoxia, C aoa and haemolymph lactate concentration had increased so that they also were not significantly different from their settled, normoxic levels, P ai Ol!, on the other hand, did not increase above its initial, hypoxic level (Fig. 7). On one or two occasions the E.c.G. wires broke during an experiment, and the necessary handling of the animal in order to attach new wires caused a large rise in heart rate which took several hours to return to its previous level. For example in one lobster, heart rate 12 h after the induction of hypoxia was 27 beats min" 1. New E.C.G. wires had to be replaced 4 h later, and immediately after this procedure, heart rate was at 74 beats min" 1. As long as 20 h after this handling, heart rate was still at 44 beats min" 1 and not until 12 h after this, had heart rate fallen to 32 beats min" 1. These artifically high values of heart rate caused by handling are not included in the mean values which are given in the tables and graphs. After approximately 72 h at a P/,o 2 of 50-55 mmhg, heart beat was regular and the rate was 85 % of the settled normoxic value. Scaphognathite activity was predominantly bilateral with unilateral ventilation occupying 28 % of the time and respiratory pauses occurring for 6% of the time (Fig. 4). Mean values of the measured variables at this time are given in Table 1.

142 P. J. BUTLER, E. W. TAYLOR AND B. R. MCMAHON Table i. Mean values ± S.E. of the measured variables in lobsters during normoxia 48 h after being placed in experiment chamber, and72 h after exposure to hypoxia.the number of observations, n, is given with number of animals in parentheses 48 h after placement in 72 h after exposure to experimental chamber hypoxia P;,o, (mmhg) I3O-I3S SO-SS P,,o a (mmhg), n = 5 ( 5 ) 49±i3 J 5±2 C..o 2 (vol. %), n = 5 (5) O-O2±O'O8 o-68±o-n pha, n = 5 (5) 7-818 ±0-02 7-87510-044 Blood lactate (mg %), n = 5 (5) i2-9±o-7 10-3 ±1-3 Oxygen uptake (ml kg" 1 min" 1 S.T.P.D.) 0-387 + 0-024, n = 30 (12) o - 434±o-ois, n = 60 (12) Ventilation volume (ml kg" 1 min" 1 ) 233+49, «= 8 (4) 398 + 26, n = 24 (4) % Ext. 41 ± 6-6, n = 8 (4) 49 ± 1-7, n = 24 (4) Heart rate (beats min" 1 ), n = 48 (6) 33 + 2 28 ± 2-5 Return to normoxia Upon being returned to normoxic water, P a Oa increased significantly to its original normoxic level, C a> Ol!, pha, blood lactate, heart rate and % Ext. did not change significantly, although heart beat became irregular (Figs. 2, 3, 4, 7). Ventilation volume fell to its settled normoxic rate and associated with this, there was a switch to almost continual (84%) unilateral ventilation (Figs. 3, 4, 6). Immediately upon return to normoxia, P r Ot increased significantly but within 24 h it also had returned to a value which was not significantly different from its settled, normoxic rate (Fig. 3). DISCUSSION Lobsters, like crayfish (McMahon et al. 1974), take up to 48 h to settle completely in an experimental chamber under normoxic conditions, even when unrestrained. During that time, oxygen uptake and ventilation volume fall and percentage extraction of O 2 from the water increases. Oxygen uptake in the settled lobsters was similar to that recorded by Spoek (1974) for the same species after they had been left for at least 3 days in the experimental apparatus. The oxygen requirements of the settled lobsters were met by intermittent use of the respiratory and cardiac pumps, in fact the respiratory pump, when being used, was predominantly active on one side only. Respiratory pauses and bradycardia under resting, normoxic conditions have been reported previously in lobsters (McMahon & Wilkens, 1972) and in the shore crab (Taylor et al. 1973). Associated with the respiratory pauses in both animals there are reductions in P a 0>, and in the lobster, H. americanus, McMahon & Wilkens (1972) found that P Ot Oa could get as low as 20 mmhg during such pauses, whereas during ventilation it was as high as 60 mmhg. A similar range of P a< Oa values was recorded in settled, normoxic lobsters in the present study, which would indicate that some haemolymph samples were taken during a respiratory pause while others were taken from animals that were ventilating fully. These periodic respiratory pauses, bradycardia, and oscillating P a Oi in the settled, normoxic lobster, are strikingly similar to the situation that has been reported to occur in aquatir. vertebrates, such as the Port Jackson shark (Capra, 1976) and in the other poikilothermic vertebrates, amphibians and reptiles. Under resting conditions amphibians (de Jongh & Gans, 1969; West & Jones, 1975) and aquatic and terrestrial

Long term hypoxia in the lobster 143 reptiles (Boyer, 1967; Gaunt & Gans, 1969; Burggren, 1976; Gans & Clark, 1976) often do not ventilate their lungs in a continuous, rhythmic manner. Apnoeic periods are often associated with diving and under these conditions it has been found that bradycardia and a reduction in P Oi Oj occur, with heart rate and P Oj o> increasing when ventilation recommences (Jones & Shelton, 1964; Lenfant et al. 1970; Emelio & Shelton, 1974; Burggren, 1975). A point of interest is that the P a> Oj at which Xenopus (Emelio & Shelton, 1974) or Chelys (Lenfant et al. 1970) emerge is around the shoulder of the O 2 equilibrium curve for their respective blood pigments. The lowest P Oj Ol recorded by McMahon & Wilkens (1972) in the lobster during a respiratory pause is also near the shoulder of the equilibrium curve for its haemocyanin. Lenfant et al. (1970) produced evidence to suggest that voluntary breath holding in Chelys is related to P a,o 2. so it is possible that a similar mechanism is present in the lobster. The presence of an internal oxygen receptor in the lobster is certainly consistent with the conclusion of McMahon & Wilkens (1975). There is also strong evidence to suggest that in both decapodan crustaceans (Wilkens, Wilkens & McMahon, 1974) and in chelonian reptiles (Burggren, 1976), the close relationship between respiratory activity and heart rate may result from the interaction between respiratory and cardiac motor neurones within the C.N.S. In the present experiments, inspired P Oa was reduced to a level which took P a Qi below the lowest values recorded by McMahon & Wilkens (1972) during respiratory pauses in normoxic lobsters, and which in lobsters that had only been left to settle in the apparatus for 2 h, caused a substantial bradycardia and a clear reduction in l r Oa (McMahon & Wilkens, 1975). This value of P a> o s (approximately 15 mmhg) was obtained at a P It Oa of 50-55 mmhg and the most obvious responses of the lobsters to this level of hypoxia were in the activities of the respiratory and cardiac pumps. Both scaphognathites became continuously active and heart beat was regular, even if, on average, a little slower than during normoxia. The rise in pha associated with the hyperventilation during the initial stages of hypoxia caused an increase in the O 2 affinity of the Hey. This change in ph may, therefore, fulfil the same function as the fall in organic phosphates in the red corpuscles of eels during prolonged exposure to hypoxia (Wood & Johansen, 1973). In the lobsters, pha declined throughout the period of hypoxia, and part of this decline may be associated with the reduction in V g during the latter stages of hypoxia. It is also possible that some of the decrease could be attributed to the repeated sampling of the haemolymph (Truchot, 1975). Certainly it has been shown that handling does cause acidosisin lobsters (McMahon, Butler & Taylor, 1978). Thus, under more natural conditions, it may be inferred that pha declines below the initial high value seen soon after exposure to hypoxia, but remains above the settled normoxic value, thereby maintaining at least a slight increase in the O 2 affinity of the Hey (McMahon et al. 1978). Whether or not there were changes in haemolymph ionic concentrations during hypoxia which could have affected the P 50 of the Hey (Spoek, 1967) was not determined. The oxygen carrying capacity of eel blood rises quite considerably during prolonged exposure to hypoxia (Wood & Johansen, 1972), but there was no evidence from the values of C a> Oj upon return to normoxia that Hey concentration had increased significantly in the lobsters after exposure to hypoxia for 120 h. This may have been related to the fact that they were not fed during the experiments.

144 p - J- BUTLER, E. W. TAYLOR AND B. R. MCMAHON Thus at the level of hypoxia that they experienced in the present investigation, lobsters that had previously been left to settle completely in the apparatus maintained their oxygen uptake merely by maintaining a regular heart beat and increasing ventilation volume. The latter was achieved, at least partly, by almost continual bilateral ventilation and virtual abolition of the respiratory pauses. In addition, there was an initial large increase in the O 2 affinity of the Hey and most likely a reduction in venous oxygen content (McMahon & Wilkens, 1975; Taylor, 1976). The maintenance of V o% during hypoxia together with the lack of accumulation of lactic acid, would tend to indicate that there was no anaerobiosis during the period of hypoxia. Although lobsters do accumulate lactate when exposed to severe hypoxia (Spoek, 1974), it should be remembered that, amongst invertebrates in particular, the absence of lactate accumulation does not necessarily mean that there is no anaerobiosis (Hochachka & Somero, 1973). Indeed, there was a marked increase in V o% when the lobsters were returned to normoxic water and although there may have been replenishment of haemolymph oxygen content (cf. Jones, 1972), this could not account for the whole of the observed rise in V Ot. Assuming a haemolymph volume of 300 ml for a 1 kg lobster (Belman, 1975) and if haemolymph oxygen content fell by 0-5 vol. % during hypoxia, then it would require a maximum of 1-5 ml O 2 to restore the O 2 in the haemolymph. Oxygen uptake upon return to normoxia was approximately o-i ml kg" 1 min" 1 higher than during hypoxia, so at this level it would have taken 15 min to replace the haemolymph O 2 reserve. The elevated level of X? Ol mentioned above was recorded some 2 h after P 7j Oj had been increased. Possible explanations to account for the extra O 2 uptake upon return to normoxia are that the lobsters become more active, although we have no evidence that this was so, or that there was some anaerobiosis during the period of hypoxia, which involved the succinate pathway. There is evidence to suggest in Mytilus edulis that both aerobic and anaerobic pathways function simultaneously during hypoxia, that different tissues can respond differently, that even in animals acclimated to low P o% the anaerobic pathway is still operative (maybe as a result of the increased energy cost of ventilation) and that subsequent removal of the end products of the succinate pathway can manifest itself as repayment of an oxygen debt (Bayne & Livingstone, 1977; Livingstone & Bayne, 1977). Although we could find nothing in the literature to suggest that the succinate pathway operates in crustaceans, if the assumption is made that it does, then on the basis of the above results from Mytilus, the rise in V o^ upon return to hypoxia could be accounted for as follows: as ventilation volume increased during hypoxia the O 2 demand of the respiratory muscles would also have risen, and yet the oxygen uptake of the whole animal did not rise initially; thus it is possible that because of the low P a Oj, there was some anaerobiosis, perhaps in some more deep lying tissues, during hypoxia, and that the succinate pathway was involved; upon return to normoxia the non-lactate end products of anaerobiosis were then oxidized. Additional circumstantial evidence in favour of this possibility is the fact that haemolymph CO, levels increased during the period of moderate hypoxia in the lobster, despite the hyperventilation (McMahon et al. 1978). Carbon dioxide may be generated by the succinate pathway during anaerobic metabolism (Hochachka & Somero, 1973).

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