a study of the effect of temperature changes...

J. Exp. Biol. (1970), 5*. 177-192 I 7 7With 9 text-figuresPrinted in Great Britain

A STUDY OF THEEFFECT OF TEMPERATURE CHANGES ON THE

RESPIRATORY PUMPS OF THE RAINBOW TROUT

BY G. M. HUGHES AND J. L. ROBERTS*

Department of Zoology, Bristol University

(Received 18 August 1969)

The pumping mechanism maintaining water flow across the gills has been investi-gated in the trout using pressure transducers and cine analysis (Hughes & Shelton,1957, 1958). It was shown that the mechanism could be considered as being madeup of a buccal pressure pump and opercular suction pumps, the two being separatedfunctionally by the gill resistance. In this species, Salmo gairdneri, the relative con-tribution of the two pumps appeared more or less equal, as judged by the areas of thecorresponding phases of the differential pressure across the gills. Further studies ofthe respiratory pumps with special reference to their muscular basis were madeusing electromyography (Ballintijn & Hughes, 1965) and emphasized the manycouplings between the two pumps. Although several studies have been made on theinfluence of environmental changes on gas exchange (Randall, Holeton & Stevens,1967), no detailed account has been published on the modifications that occur in gillventilation mechanisms and interactions with cardiac cycles when trout are subjectedto stress. The present series of experiments forms part of such a study in which electro-cardiograms were obtained simultaneously with electrical recordings from parts ofthe respiratory neurones in the medulla, from some respiratory muscles, and withpressure recordings from the buccal and opercular cavities. All were carried outduring subjection of rainbow trout to warming thermal stress. A preliminary reportof this work has already been given (Roberts & Hughes, 1967).

MATERIALS AND METHODS

The rainbow trout, Salmo gairdneri, used in these experiments were obtainedregularly from a hatchery at Nailsworth, Gloucestershire. Specimens were usually of200-300 g. and were allowed to acclimate in holding tanks within the laboratory forat least 7 days at a temperature of 15°+ i° C, but in some instances the acclimationtemperature was 180 ± i° C. (Fish / ) .

Each fish was anaesthetized in MS 222 (o-i g./l.) before it was fixed in the experi-mental tank and was allowed to partially recover to a lighter level of anaesthesia afterthe electrodes and/or pressure needles had been placed in position. The fish was thensecured lightly in a body clamp of a sling type (velcro plush and hooklet bandI in. wide), but more rigidly in a second clamp attached across the supra-orbitalridges of the frontal bones (Fig. iB). The water in the experimental tank formed part

• Present address: Department of Zoology, University of Massachusetts, Amherst, Mass., U.S.A.12 E X B 52

i 7 8 G. M. HUGHES AND J. L. ROBERTS

ThermoregulatedI circulation T

Fifi T C a l - Fin©—Bath 7©

Bath

Opercular(differential)

Bath

e-8

s v

Buccal

Partitionblock

Fig. i. Diagrams of the experimental tank and arrangement for recording ventilatory pressuresof trout during temperature changes.

A. Section of tank showing water circulation and position of recording needles and cannulae.The experimental tank and partition were made of } in. plywood treated with polyurethanevarnish and are shown cross-hatched.

B. Transverse section to show the fish held in its sling and clamped across the supraorbitalridges.

C. Diagram of'plumbing' used with the two 268 B differential manometers in these experi-ments. Taps are shown in a position for recording the opercular and buccal pressures separately.Tap 5 is turned through 180°, Tap 3 through 4s 0 for recording the differential and buccalpressures.

Temperature and the respiratory pumps of trout 179

of a closed circuit 5 1. in volume (Fig. iA). Circulation was maintained by an air-liftdevice at a rate sufficient to allow accurate temperature control as well as systematicvariation of the temperature (i° C. change/3 min.). The air-lift circulation also servedto maintain the dissolved oxygen content of the bath water at saturation regardless oftemperature (O2 content determined by Winkler). Pressure changes were recordedthrough hypodermic needles (o.D. = 0-83 mm.) with holes in the side (Fig. 1), whichwere fitted on to lengths of lead tubing attached to Sanborn 268 B differential pressuretransducers. In most experiments an arrangement (Fig. iC) was made for the simul-taneous recording of either the buccal and opercular pressures or the buccal anddifferential pressures.

10 20 30 40

15 -

50

Fig. 2. Graphs showing the relation between water bath (—) and brain ( • - - • ) temperaturesof 7 trout lightly anaesthetized and held in the apparatus shown in Fig. i. The temperatureswere taken at given times during warming of the water bath at a rate of i° C./3 min. Braintemperature increase lags behind water temperature. The curves can be used for interconversionof water and brain temperatures given in the tables and text figures of this paper.

In a typical experiment the fish was allowed to settle in the experimental tank forabout 1 hr. at its acclimation temperature and under an anaesthetic level which keptthe fish generally quiet but which did not block gentle sculling movements ofthe pectoral fins (50-60 mg. MS 222/L). Recordings were made of the buccal,opercular and differential pressures, electrocardiograms and any other parametersused for that particular experiment. The water temperature was then gradually in-creased and recordings were made at appropriate times. Temperatures were notallowed to rise very much above a bath temperature of 290 C. before being returnedto the original level at the same rate of cooling as for heating. In only a few experi-ments, however, was complete recovery possible once the brain temperature had been

180 G. M. HUGHES AND J. L. ROBERTS

above 270 C. The warming rate of i° C./3 min. was chosen to conform with warmingrates frequently used in the determinations of lethal thermal limits (Fry, 1967) andto minimize possible thermal shock effects (faster warming) and significant thermaladaptations (slower warming). As a result, the deep body temperature was found tolag behind the increase in bath temperature during warming. Body temperatureswere taken by means of a bead thermistor implanted in the cranial cavity so that thetemperature recorded at any one moment most nearly matched that of the respiratoryneurones in the medulla. Figure 2 was constructed in order to permit intercon-versions of temperatures at any time during a warming sequence and is based onparallel recording of water bath and cranial temperatures. Except where otherwisenoted, all temperatures given in this paper are those of the water bath.

RESULTS

A. The typical differential pressure record

In the original work on trout (Hughes & Shelton 1957, 1958), only a single con-denser manometer was available and it was necessary to superimpose successiverecordings of the buccal and opercular curves in order to obtain the differential

Opercular movement

Abduction

Bucca!

Seconds

Fig. 3. Rainbow trout. A. Simultaneous recording of the opercular movements and pressurechanges in the respiratory cavities. Initially the buccal and opercular pressures are recordedand then the buccal and differential pressure across the gills. The usual convention is adoptedfor the latter to be positive when the pressure in the buccal cavity exceeds that in the opercularcavity. Zero in all cases is the pressure outside the fish.

B. Differential pressure recordings from an individual trout during warming at the tempera-tures shown. Notice the appearance of double reversals at 26-2° and 29-2° C.


BFessure from them. With the development of the liquid differential transducer it hasbecome possible to record the differential pressure directly. Hence a series of experi-ments was carried out for this purpose and, as well, to compare such recordings withthose obtained using the cannulation technique of Saunders (1961). It was found thatdifferential curves of the type derived by Hughes and Shelton were the most common(Fig. 4 A) with both methods of recording but details such as the relative balance ofthe buccal and opercular phases varied more as between specimens than as between

1.4-9° C.

17-1° C.

22 0° C

255° C. 270° C.

1 sec.

Fig. 4. A. Tracings from simultaneous recordings of the buccal and opercular pressures showntogether with the derived differential pressure as used by Hughes & Shelton (1958). Forcomparison, a tracing from the differential pressure recorded a few moments later is shownbelow.

B. The differential pressures recorded during experiments in which the trout was subjected totemperature stress showing the change in area of the three main phases. During phase a theopercular pump predominates and in b, the buccal pump; c is the reversal phase. Numbersrefer to the areas of the three phases. In this particular trout the opercular phase (a) wasgenerally the largest.

methods. Unlike the results obtained in earlier experiments, subsequent recordingsusing rainbow trout from the same hatchery have shown periodic 'coughs' especiallyduring early stages of their acclimation to an experimental situation. Recordings ofsuch coughs were obtained during electromyographic studies and more recent re-cordings have been obtained in experiments on suspended solid pollutants (Hughes,1970).

Typical recordings are shown in Fig. 3. The buccal pressure pump and oper-cular suction pump phases can easily be distinguished and are separated by two transi-tional phases, one of which shows a clearly defined reversal. Similar recordings with


the animal restrained in the clamps were obtained using either the cannulation technique or via the pressure needles. The same was true of the pressure changes recordedduring the coughs so that any differences which appear in experiments with restrainedfish and fish relatively unrestrained in a closed circulation bath cannot be ascribed tothe recording method.

For the more detailed analysis of the pressure waveforms recorded in this way,the following methods were used: (i) the total amplitude of the opercular or buccalpressure was measured directly from the recordings; (ii) the differential record wasdivided into three parts as shown in Fig. 4B; during two of these (a, b) the buccalpressure exceeds that in the opercular cavity and the third (c) is formed by thereversal pressure. Under certain conditions the second transitional phase also showsa reversal and this will be referred to as a double reversal. From tracings of therecords measurements were made of the area of these different phases, so as to givesome indication of the relative contribution made by different parts of the respiratorycycle to the pressure gradient operating across the gill resistance to produce the waterflow. A relationship between the differential pressure and ventilation volume has beendemonstrated (Hughes & Shelton, 1958; Hughes & Saunders, 1970). Thealgebraic sum of the area beneath the differential curve multiplied by the frequency/min. gives a differential pressure equivalent for the minute volume and is thereforea useful measure of the performance of the pumping mechanism during varyingconditions, especially when these produce frequency change. The mean differentialpressure is obtained when this equivalent [(a + b —c) x frequency] is divided by timeexpressed in the same units as for the differential area measurements.

B. The effect of temperature stress

The frequency of respiration. In all preparations it was found that an increase intemperature led to an increased frequency of pumping. There was some variation inthe details of this relationship but a number of preparations suggested that the rateof rise in frequency steepened as the temperature reached about 240 C. This is theequivalent of a brain temperature (deep body or core temperature) close to 220 C. atthe warming rate used.

Table 1 shows the average frequency of respiration at 160 and 260 C. for a number ofpreparations as calculated from the pressure recordings. The data of Fig. 5 is from anexperimental series in which electrical activity of respiratory neurones in the medulla

Table 1. Frequencies of respiratory movements at water temperatures of 160 and 260 C.for 7 individual trout. Notice variation in both absolute values and percentage increases

FishFGIJKLM

Means

i6°C.120/min9482

n o75

IOS

" 5IOO-I

26° C.160/min198116165150180300

1812

Differences

4 0104

34557575

1858 1 1

0//o

33i n

4150

100

7 i160

80

Mean percentage change = 80-9%, hence average overall Qlo = I-8I (range i-33-2-6o).


fcvas recorded. Both sets of data refer to changes in frequency which occurred as theWater temperature increased. They show that there is a sizeable individual variationnot only in the base frequency level at the commencement of the experiment but alsoin the percentage increase. This suggests, as do other characteristics of this and otherdata, that there may be a variety of ways in which a fish compensates for stress, e.g.changes in stroke volume can occur and alter minute volume even at a relatively con-stant frequency. The fact that the incremental change in ventilation rates as shown

400

2001-

c

I8. 100

| 80

* 60

14

-

X-t-A

i i

—f—

i i i

Ventilation

• j -4-

Heart

i i i i i

r

i i i • i

16 18 20 22 24 26 28 30

Water temperature (°C.)

Fig. 5. Plots showing the effect of wanning the water bath (i°/3 min.) on the frequencies ofventilation (• • ) and heart ( x x ) beat of seven trout. Vertical bars above and belowthe mean values represent ± 2 standard errors of the means. Qw for the mean values is asfollows:

15-21° 21-26° 26-28° OverallVentilationHeart

118207

a-34149

4-02 1721-76(15-26°)

in Fig. 5 was found to be very small, between 15° C. and 21° C. (Q10 = 1*18) is one typeof evidence. Another piece of evidence was the observed sharp rate change above2i° C. which may represent the upper limit to such other means of compensationand/or reflect the onset of reflex cardiac inhibition that becomes noticeable above210 C. Value of J io at the higher ranges were 2-34 between 21 ° and 26° C. and 4-02between 260 and 280 C. The calculated overall Q10 values found for the experimentsummarized in Fig. 5 and Table 1, were only 172 and I-8I respectively. Clearly severalinteracting phenomena are involved.

In those experiments in which the readings were continued as the temperature wasreduced to normal levels it was found that the respiratory frequency was higher fora given temperature than during temperature increase (Fig. 6). This was partly due tothe fact that with the warming and cooling sequence used (i°C./3min.) thermalgradients develop between bath temperatures and brain temperatures of the fish.Another factor involved in the hysteresis might be due to the paying-off of an oxygendebt incurred during the temperature rise.


Effects on cardiac frequency. The same slow temperature rise between 150 and 210 Cresulted in cardiac rate changes with a Q10 of 2-07. Between 21 ° and 26°C. the rateof change in cardiac frequency declined (Q10 —1-49). Very marked deceleration in rates(negative temperature coefficients) occurred above 260 C. up to the maximum per-mitted (29°~3O° C). It was also at this 'break point' in temperature that the sharprise in ventilation frequency was found to occur with further warming (Fig. 5).

II

250

| | - 1 9 0

I g 160Sg-130* £ 100

70

v3

Q.

E

_ O•Si"

£s

4

3

1 -

1 1 1 1

~*^*\ 1 1

- / ^ ^

/- Buccal

pressure1 1 1 1

: ^

i i i i

j * j» _

i i i i

i/\ \ppercular\ \ pressure

A \ \

r X J »v1 1 1 1

1 1 1 1

14 16 18 20 22 24 26 28


14 16 18 20 22 24 26


28

Fig. 6. Effect of change in bath temperature on the respiratory pumps of two individual fish.A. A rise in respiratory requency is accentuated above 22-23° C. Note hysteresis as tempera-

ture is lowered (O O).B. Amplitudes of the buccal (A- • ) and opercular ( • • ) pressure waveforms

showing more marked effect on the latter. During recovery (A, O) the buccal pressure wasespecially influenced in preparation 1.

C. The mean differential pressure increased and then fell above 24° and 200 C. in the twocases.

Possibly this serves as a means for adjustment to the parallel decline in cardiacfrequency. The ratios between cardiac and opercular frequencies determined fromthe data of Fig. 5 changed from 1:1-7 at 150 C. to 1:1-2 or nearly equal rates at 210 C.The ratio returned again to 1:1-7 at 27° C. and finally reached 1:3 as the high-temperature bradycardia developed. Whether or not increasing stress tends to in-crease the degree of cardio-ventilatory coupling was not always obvious for reasonsof experimental design and methods of recording. For example, the temperature riseused was continuous at 1° C./3 min., a procedure which precluded the accumulationof sufficient data at any given temperature. A tendency to phase-lock or synchronizeover the temperature range when the two activities were similar (220 and 26-5° C.)


was not evident in the data used for the preparations of Fig. 5, but in other cases therewere indications that it occurred.

Abnormalities in the electrocardiogram, such as arrhythmias, prolongation inP-R intervals and dissociations of atrial and ventricular beats, were not found to occuruntil very high brain temperatures had been reached (above 250 C.) and progressivefailure in ventilation became obvious. The latter was usually identified by a rapid risein the incidence of atypical respiratory movements of a spasmodic nature at about26° C. Generally the cardiac abnormalities were not seen until regular ventilationcycles had ceased and when hypoxia was likely to be severe.

Effect on the buccal and opercular pressure. An increase in temperature was accom-panied by significant increases in the amplitude of both the buccal and opercularpressure waveforms (Fig. 6) which tended to be more marked for the opercularpressure in most preparations. At the higher temperatures (above 23° C.) the pressureamplitudes fall, that of the buccal pressure begins at a slightly lower temperature thanthe opercular pressure. During recovery the opercular pressure does not have such agreat amplitude at a given temperature as during the warming period (Fig. da, b).Such differences in amplitude between the increasing and decreasing phase of tempera-ture changes are not usually so clear for the buccal pressure. During recovery thebuccal curve may or may not be greater in amplitude than during stress and seems tochange relatively more than the opercular pressure during this period. In some casesthe buccal pressure increased in amplitude to a far greater extent during recovery thandid the opercular pressure (Fig. 61).

Apart from increases in amplitude the increase in water temperature may also giverise to changes in form. At intermediate temperatures the pressure waveforms gener-ally become more regular and constant in form, but above temperatures of 21-22° C ,the buccal waveform sometimes shows an increase in its negative phase which may beassociated with a fall in gill resistance and consequently a greater effect of opercularsuction on the pressure within the buccal cavity. There are also frequent abnormali-ties, such as superimposed biphasic waveforms, which suggest alterations in thecoupling between the two pumps, especially as they are accompanied by the appear-ance of double reversals in the differential curves.

It is difficult to make overall generalizations regarding pressure waveforms, for notonly do they vary under normal conditions, but, because of the relatively balanced roleof the two pumps in the trout, there are several apparently equally effective ways inwhich they can respond to the temperature stress. The examples plotted in Fig. 61and II illustrate this variability.

Effect on the differential pressure curves. There tend to be effects on the opercularsuction and buccal pressure pump phases of the differential curve which are relatedto changes in amplitude of the individual waveforms. The transitional phases fre-quently became more marked, and this was especially true of the reversal phase innearly all preparations. As the temperature was raised, there was an increase in theproportion of the respiratory cycle during which the respiratory pressure showed areversal. This varied; for example, the percentage reversal for fish G was 7-1% at15° C, rising to 10% at 22-5° C. and 28% at 25-5° C. At the higher water tempera-tures there was often a double reversal which contributed to the greater total reversaltime. In fish /, however, which was generally a good preparation with well balanced


buccal and opercular pressures, at 15-6° C. the reversal time was 12-5% but it hadfallen to 9 7 % at 23-5° C. However, at 26-4° C. there was a 25% reversal time withdouble reversals. On cooling the same fish, double reversals fell out when thetemperature had fallen to 230 C. This fish survived a water temperature of 22*9° C.In other fish the reversal time at normal temperatures (150 C.) might be as high as29%. In this one case (fish L) the reversal time with warming rose to 39% at 21-5° C ,but then dropped until the stress temperature approached lethal (about 290 C.) and'coughing' began. There were instances during the warming stress when for short

16

14

12

10

8

6

4

2

15 20 25 30 10 15 20 25 30

Water temperature (°Q

Fig. 7. Effect of increases in water temperature on the differential pressure curve. The areas(arbitrary units) of the three components (a, b and c) of the differential pressure are plottedindividually. In one fish (7) the buccal pump (6) is the larger phase at 15-25° C , but in fish Jthe opercular pump is more important.

periods the pumping efficiency appeared to increase, as in fish L at 25 -3° C. when thereversal time dropped to 5*7% and no longer showed double reversals. It is worthyof note that the buccal pump was dominant in this fish throughout; such inequalitiesbetween the pumps seem to increase the occurrence of double reversals.

It was found that there was usually an increase in the area beneath the differentialpressure curve considered in relation to a single cycle or on an overall basis. Thedifferential area/stroke (a+b—c) usually increased with temperature and then de-clined as it reached higher levels (about 210 C.) as illustrated for fishes / and J inFig. 7.

There was also a tendency for the total differential area/unit time or the meandifferential pressure (Fig. 6C) to increase as the frequency of respiration in-


creased, but at higher temperatures (and frequencies) there was once more a declinecoupled with the increased double reversal phases mentioned above.

Under conditions of greatest temperature stress the fish makes very rapid pumpingmovements of small amplitude and probably pumps a relatively small total volume ofwater across the gills. At intermediate temperature stresses there were indicationsthat the fish was compensating by increased levels and efficiency of pumping. One

15

13

a.« 9

14 16 18 20 22 24 26


28 30

Fig. 8. Relationship between area (arbitrary units) beneath the differential pressure curve andwater temperature in 4 individuals as the water they were breathing was warmed. There is atendency for the level of this pressure to be maintained or slightly increased and then fall at aparticular temperature which varies from preparation to preparation.

sign of this was taken to be the relative stability of the mean pressure below tempera-tures considered to be severely stressful, i.e. about 26° C. (as shown, for example, byfish /, etc.) (Figs. 6 and 8). Also the variation found for the patterns of change in thepressure curves with warming was considerable among the seven trout used in thispart of the study. This suggests that differing combinations of frequency and ampli-tude are utilized by individual fish in the operation of the ventilatory pumps. Althoughit is obvious that in any one fish, buccal and opercular pumps cannot vary separatelyin frequency, pressures developed during ventilation can and do differ greatly asbetween the two cavities. This means that for any one fish the energy needed to passwater through the gills can be expended equally by both pumps or predominantly by


one of them. Figure 7 illustrates two cases. In trout / the opercular pump dominateduntil pumping efficiency fell to very low levels at about 23 ° C. (CT). In trout J thebuccal pump dominated for the entire warming period. These results confirm thoseobtained from measurements of the amplitude of the individual pressure waveforms.The declines in differential pressures shown by these two fish with severe thermalstress were also found with the other animals in the experimental series (Fig. 8).Because the pressure changes have such low amplitudes at higher temperatures (e.g.Fig. 3B and 4B), and may also change in form, it was difficult to separate the twophases generally ascribed to the buccal and opercular pumps. The independence ofthe two components of the differential curve may be greater in the trout than in otherfish because of the lesser degree of coupling between the two pumps as has beenindicated in the recent comparison made by Ballintijn (1969) of the carp and trout.

DISCUSSION

When a fish is subjected to thermal stress as in the present series of experiments, theresponse of the respiratory and cardiovascular systems is complex because of thevaried effect of the environmental change. There is an overall effect on the metaboliclevel, as with terrestrial animals, and such studies have been carried out for brooktrout by Job (1955), and Beamish (1964). It is noticeable (Fig. 9) that the Q10 ofoxygen uptake declines at higher temperatures, an effect similar to that which wasnoted by Rao & Bullock (1954) for a wide variety of fish and other poikilotherms. In

Table 2. Effects of temperature upon the standard O2 consumption of brook trout andsome physical characteristics of fresh-water environments

Temperature °C.

1. Oxygen uptake'1'"Salvelinus fontinalis

2. Density of water"1

3. Viscosity of water"'

4. Diffusion rate141

5. Absorption coefficient"*

6. Permeation coefficient

(calculated from 4 and5 above)

ml./kg./hr.%010

Centipoises%

0 i .'D' (cm.l/sec. x 106)%

0,0'<x'(ml. O,/ml. H,O,

760 mm, Po.)%

0 i .D' = axD(x iol)0//o

S

2 3 82 9 8

4-2

09999

1-521 0 0

1 4 66 2 2

00429

1 0 0

6 2 6

9 4 2

1 0

47-459-2

2-

09997

I-3I86-1

1

1-6S

7 0 2

1

0-0380

88-6

1

6 2 7

9 4 4

15

6 8 98 6 2

1 1-4099911 4 1

75 0

•31 (overall)

1*5788

•27 (overall)

0-0342

7 9 8

•23 (overall)633

953

20

8OOIOO

09982I-OO

6 5 8

2 0 8

885

0-0310

7 2 3

6-4597-1

25

—

0997c

0 8 958-6

2 3 5IOO

0-0283

6 6 0

6-65

IOO

0,0 1-05 (overall)

Based on: (1) Job (1955); <«> Beamish (1964); <»> Lange (1961); <«> Himmelblau (1964).


pir-breathing animals the direct effect of temperature changes on the respiratoryenvironment is not very significant, but for an aquatic animal there is a very markedeffect on important physical characteristics of the water. These include its viscosityand density, and from the point of view of gas exchange, the solubility and diffusionrate of oxygen in water. Changes in these parameters with temperature increasesbetween 50 and 25 ° C. are summarized in Table 2 and Fig. 9.

100

90

80

60

50

30 -

10 15 20

Water temperature (°C.)25

Fig. 9. Graphical representation of the effect of temperature increase on physical features ofimportance in respiration of fresh-water fish. Increase in the oxygen consumption is alsoshown. The different parameters are numbered as in Table 2.

The lowering of viscosity must certainly reduce gill resistance and ventilatory workand thus partially compensate for the increased ventilation necessitated by the decreasein oxygen content resulting from a fall in solubility (Hughes, 1963). Yet, becausethe percentage utilization typically falls with increased flow (Saunders, 1961; Hughes& Shelton, 1962), this advantage may be effectively masked. At first sight, the rise inoxygen diffusion rate with temperature (3 %/° C), might be expected to aid gas exchange,but this will not be so great because gas transfer involves a permeation coefficient andthat of Krogh is most often applied in such circumstances (Steen & Kruysse, 1964;Hughes, 1966). From Table 2 and Fig. 9 it can be seen that the product of a and D, whichis usually taken as equivalent toKrogh's permeation coefficient (D1) (Radford, 1964),


increases relatively slightly over this temperature range. Consequently facilita^tion of gas exchange with a rise in temperature is not so great as expected. Thus theeffect of temperature on the solubility of oxygen influences oxygen uptake adverselyin two ways: first, by reducing the effective diffusion coefficient and secondly byreducing the total oxygen content of the water and so necessitating greater ventilation.Increasing ventilation will, however, reduce the thickness of the water film over thesecondary lamella surface, which provides the major resistance to gas exchange (Hills& Hughes, 1970). It is difficult to draw up a complete balance sheet for these differentfactors but clearly the increased metabolic demand presents a considerable load forthe respiratory pumps.

The results summarized in this paper show how the fish responds by an increasenot only in the frequency of its ventilation and heart movements but also in theamplitude of pressure changes in the respiratory cavities which must result in anincreased stroke volume. Frequency is a relatively easy parameter to measure andshows significant effects of temperature on both the ventilatory and cardiac pumps.The observed effects upon heart rate were as expected (Q10 of about 2 until 210 C.)but rate changes in ventilation proved not to be so predictable. Qw values for thechange in ventilation frequency ranged from nearly 1 between 150 and 210 C , to ahigh value of 4-02 between 260 and 280 C. It is of interest that changes in strokevolume seem more important than frequency increases during the initial heat stressbecause during hypoxia frequency also increases, mainly during the more extremestresses (Hughes & Saunders, 1970). One possible interpretation of these effects isthat the initial rises in ventilation and of cardiac frequency are sufficient to provideenough oxygen to meet the raised metabolic demands, possibly by adjustments in theflow patterns of water and blood across the gills. But added stress leads to ventilationbecoming inadequate and possibly the resulting depletion of oxygen in the bloodevokes the bradycardia. This cardiac 'braking' is paralleled by a sharp rise in ventila-tion frequency, perhaps as a compensatory consequence. The inadequacies of theventilatory pumps can be partly offset, as indicated by a rise in brain POt levels, bya stream of water into the mouth (Roberts & Hughes, 1967).

The precise mechanisms mediating bradycardia are only partly understood. Itseems clear that in trout and some other teleosts the efferent pathway for bothbehaviourally and physiologically induced bradycardia is via vagal cholinergic fibreswith endings on the heart (Labat, 1966; Randall, 1968; Roberts, 1968). In somespecies the bradycardia which occurs at high temperatures is partially relieved bybilateral vagotomy (Labat, 1966) or by pre-treating the fish with pericardial injectionsof atropine (Roberts, 1968). It may be assumed that the cardio-inhibition found above260 C. in these experiments is the final result of hypoxia related to failure of the cardio-ventilatory system to maintain adequate oxygen levels at the appropriate receptors (seeRandall & Smith, 1967). If this assumption is correct in the context of the presentwork, receptor detection of oxygen must be occurring centrally (e.g. in the brain orcardiovascular system) rather than peripherally in the branchial system, for airsaturation was maintained in the water passing over the gills at all temperatures. Yetresults of Randall & Smith (1967), also with rainbow trout, contrast in that peripheraldetection probably induces the hypoxic reflex bradycardia that occurs in water withPOt less than 80-100 mm. Evidence for the functioning of both central and peripheral


Reception of POt changes controlling ventilation has been found in Callionymus(Hughes &3Ballintijn, 1968).

Still another possibility exists which seems not to have been tested, and that is thatthe bradycardia results from the input of thermal receptors which can modulate thevagomotor centres. The probability is that many afferent pathways exist, not allrelated to oxygen detection, which can induce reflex bradycardia via vagal action.This becomes more evident when consideration is given to the observation thatimmediate bradycardia results from lifting fish out of water (Serfaty & Raynaud, 1957),and as a result of visual disturbance (Labat, 1966; Roberts, 1968).

SUMMARY

1. Trout subjected to changes in water temperature (i°/3 min.) between 150 and300 C. showed a number of responses in their ventilation and cardiac mechanisms.

2. Ventilation rate increased slowly over the range i5°-2i° C , but increased morerapidly at higher temperatures (2i°-26°, Q10 = 2-34; 26°-28°, Q10 = 4-02). Cardiacfrequency fell markedly about 26° C, and this bradycardia suggests that above thistemperature the ventilation mechanism is inadequate to maintain a sufficient level ofblood Po . Possibly this insufficiency results from a failure of the pumping mechanismto increase or even maintain a large minute volume at high frequencies.

3. Pressure recordings indicate those parts of the ventilatory mechanism which aremainly involved in these responses. Increases in the buccal, opercular, and meandifferential pressures indicated that the volume of water pumped across the gillsincreased during the initial stages of warming and only at higher temperatures didfrequency become involved.

Variability in the balance between the buccal and opercular pumps among indivi-dual trout becomes even more apparent under temperature stress, as in some casesthe opercular pumps seem to be mainly involved in the increased ventilation but notalways.

4. The action of the buccal and opercular pumps seems well co-ordinated, especiallyat intermediate temperatures, but serious uncouplings occur at higher temperaturesas indicated by the shape of the pressure waveforms and especially the appearance ofdouble reversals in the differential pressure curve.

5. The relationship between cardiac and ventilatory cycles was not studied ingreat detail, but there were certainly indications of changes in coupling. However, itwas clear that the two rhythms do not always become phase-locked even at hightemperatures.

6. The effect of changing temperature on the physical environment of the fish isdiscussed in relation to these observed responses.

Equipment was provided from a Natural Environment Research Council grant toG.M.H. J.L.R. was supported by Special Fellowship GM2531 of the U.S. PublicHealth Service.


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a study of the effect of temperature changes...

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