THE RELATIVE EFFICIENCY OF GASEOUS EXCHANGE ACROSS THE LUNGS AND GILLS OF AN AFRICAN LUNGFISH PROTOPTERUS AETHIOPICUS

J. Exp. Biol. (1970), 5, 1-15 j With 1 text-figures Printed in Gret Britin THE RELATIVE EFFICIENCY OF GASEOUS EXCHANGE ACROSS THE LUNGS AND GILLS OF AN AFRICAN LUNGFISH PROTOPTERUS AETHIOPICUS BY B. R. MCMAHON Deprtment of Zoology, University of Bristol* (Received 31 Mrch 1969) Of the three gener of lungfishes living tody Neocertodus, the Austrlin lungfish, is possibly the most primitive. The respirtory physiology of this niml hs been extensively studied in recent yers (Grigg, 1965, b, c; Lenfnt, Johnsen & Grigg, 1966; Johnsen & Lenfnt, 1967) nd the conclusions these uthors drw support the ssumptions of the erlier workers in this field (Den, 1906; Longmn, 1928; Spencer, 1891), who mintined tht in this niml the lung ws n ccessory respirtory orgn used principlly when the niml ws in poorly erted wter. The gs exchnge occurring over the gills is pprently sufficient to stisfy the requirements of the niml in well-erted wter. Erlier workers on the respirtory physiology of Lepidosiren, the South Americn lungfish, suggested tht in this niml the lungs were the principl respirtory orgns (Crter & Bedle, 1930; Cunninghm, 1934; Kerr, 1897, 1898). Swy (1946) found tht the gills ccounted for only 2% of the totl oxygen uptke, nd Johnsen & Lenfnt (1967) confirmed tht the lungs were the principl site for oxygen exchnge but thought tht some crbon dioxide might be exchnged vi the qutic route. These conclusions show tht these two forms re well dpted to their predominnt hbitt. Neocertodus is usully found in permnent, well-erted wters, which only rrely become stgnnt (Grigg, 1965 c) nd is only fculttive irbrething form. Lepidosiren, on the other hnd, is normlly found in mrshy wters which re not only often extremely hypoxic nd hypercrbic, but re lso lible to dry out periodiclly. Lepidosiren is n obligtory ir-brething niml nd cn survive the periods of drought by estivting in burrow in the mud (Kerr, 1898). The hbitt of Protopterus ethiopicus is similr to tht of Lepidosiren, nd, though it hs long been ssumed tht the lungs were the principl respirtory orgn, until very recently little experimentl work hs been crried out to verify this ssumption. Prior to the completion of this mnuscript, however, severl workers hve reported studies which verify this ssumption. Jesse et l. (1968) suggest tht both lungs nd gills re importnt in respirtion in juvenile Protopterus (species not certin) but report no result from dult nimls. Johnsen & Lenfnt (1968), however, demonstrte tht the lung is the principl orgn in oxygen uptke in Protopterus ethiopicus. In this study experiments to elucidte nd quntify the ctul efficiency of lung nd gill in gseous exchnge will be described nd discussed. Present ddress: Assistnt Professor of Biology, Deprtment of Biology, The University of Clgry Albert, Cnd.! EXB 52

B. R. MCMAHON METHODS Recordings were mde of eril nd qutic respirtory frequencies, both in nimls immersed in well-erted wter with ccess to ir nd in nimls which were confined either under wter or in moist ir. The nimls were confined in Perspex observtion tnk which could be perfused with either wrmed erted wter or wrmed humidified ir. Ambient wter ws wrmed by pssge through glss wrming coil immersed in thermostticlly controlled wter bth. The temperture in these experiments ws mintined t 24+ i C. Reservoir Vlve Fig. 1. Digrm of the respirometer used in the determintion of oxygen consumption nd crbon dioxide production vi the eril nd qutic routes. A-E, tps controlling flow of ir or wter. Ventiltion rtes were recorded in three wys: () observed by the investigtor nd trnsferred immeditely to n event recorder; (b) by the electromyogrms of the respirtory muscles picked up incidentlly by ECG probes; (c) by the pressures recorded by mens of cnnule implnted in buccl nd/or operculr cvities (McMhon, 1969). In ll the experiments nimls were llowed to cclimtize to the experimentl chmber for t lest 4 hr. nd usully over 20 hr. A record of the ctivity of the nimls ws tken t ll times, but in fct cclimtized nimls usully remined t rest on the floor of the chmber, moving only to tke ir. All recordings discussed here were tken from nimls t rest. Rtes of gs exchnge were mesured by respirometry. The simultneous mesurement of eril nd qutic gs exchnge poses problems in the lungfish, s strems of respirtory ir nd wter must be kept seprte to void interdiffusion of gses.

Gseous exchnge efficiency in Protopterus ethiopicus 3 Methods where the niml is prtitioned using rubber membrnes, s used by Vn Dm (1938) nd Berg & Steen (1965), re not esily dpted for use with freeswimming niml brething ir or wter t will, nd my lso impir the respirtory mechnism (Piiper & Schumnn, 1967). In these experiments the rtes were mesured using specilly designed respirometer in which ir nd wter strems flowing pst the niml were kept seprte except t the moment of brething. This pprtus is illustrted in Fig. 1. The respirometer consisted of Perspex tube flnged t both ends which could be mounted digonlly in rigid frme. The niml ws persuded to swim into the tube nd the ends were seled by Perspex discs equipped with ' O' ring sels. The discs held number of tps which controlled the flow of respirtory medi. Once the niml ws instlled in the respirometer flow of wter ws llowed to pss through it. This wter ws collected under prffin in 1 1. smples during the experimentl periods nd ws preserved for subsequent nlysis. Air ws introduced into the respirometer only during lung ventiltion. (The niml lwys lifted the hed prior to lung ventiltion, nd on this 'signl' ir ws llowed into the respirometer). Air ws drwn in by lowering the wter level in n ccessory continer outside the respirometer but connected to it (Fig. 1). As the wter level fell in this outer continer, so it fell in the respirometer, drwing in ir from the perfusing ir strem nd llowing the niml to ventilte the lung. Immeditely fter the lung ventiltion the originl wter level in the outside continer ws restored nd the expired ir ws then forced out of the respirometer. This ir pssed into the perfusing ir strem nd ws collected in lrge bottle to wit nlysis. All gs nd wter interphses, with the exception of tht in the respirometer itself, were protected with prffin. The wter smples were nlysed for oxygen content using the Winkler method nd for crbon dioxide content by the nomogrph method of Dye (1951). The gs smples were nlysed for both oxygen nd crbon dioxide content using Scholnder \ ml. Gs Anlyser (Scholnder, 1947). All experiments were crried out in constnt temperture room (24 ± 1 C), thus ensuring tht gs nd wter smples were t the sme temperture. It ws not possible to cclimtize the nimls in the respirometer for long periods becuse continul mnipultion by the opertor ws necessry to mintin the nimls in the chmber. In fct, they were to some degree pre-cclimtized to the tubes (which were often inhbited by them when left in the 'home' tnks). This suggests tht the nimls were not seriously disturbed by their close confinement during the experimentl periods. Once instlled nd settled into the chmber the nimls very rrely showed ny ctivity not ssocited with ir brething. Smples of pulmonry gs, nd both inhlnt nd exhlnt brnchil wter were obtined from chroniclly implnted cnnule. The pulmonry cnnul ws inserted into the nterior medin sc of the lung, nd the other cnnule into the buccl nd operculr cvities s in McMhon (1969). Smples of 0-5-1-o ml. of pulmonry gs were withdrwn into syringe, the ded spce of which wsfilledwith solution which ws non-bsorbent for gses. A volume of gs equivlent to the cnnul volume ws withdrwn nd rejected before ech smple. Smples were tken t intervls through number of ir-brething cycles nd stored immersed in cooled, non-bsorbent solution for periods of 2-4 hr. to wit nlysis. Tests indicted tht no significnt

4 B. R. MCMAHON chnge occurred in smples stored in this wy. In some of the experiments the P O of the smples ws tken on withdrwl nd this provided check on the subsequent nlysis. Wter smples were withdrwn from the buccl nd operculr cvities to determine the extent of the gs exchnge ocurring over the gill surfce. During slow brnchil irrigtion (less thn i/min.) very smll smples (o-2-o'3 ml.) were tken into syringe ttched to the cnnul. The smples were tken very slowly to void drwing wter over the gills rtificilly, nd were withdrwn t vrying intervls fter the lst brnchil respirtory movement. During fster brnchil irrigtion lrger smples (0-5-1'5 c.c.) were withdrwn over period covering severl brnchil respirtory movements (0-5-1-5 min.). In ll cses volume equivlent to the cnnul volume ws tken nd discrded before smple ws tken. The smples were nlysed for oxygen nd crbon dioxide tension using n 'Eschweiler' gs nlyser. The electrodes were clibrted with gs mixtures of known oxygen nd crbon dioxide tension severl times during ech experiment. The oxygen electrode system ws very stble nd gve repetble results t 1 % level of ccurcy. The crbon dioxide electrode, however, hd slow response time t this temperture nd ws lible to drift from the clibrtion settings. These fctors my hve introduced level of inccurcy (< 5 %) into the results. RESULTS In the first series of experiments the frequencies of brnchil irrigtion nd lung ventiltion were monitored in nimls before, during nd fter confinement in either ir or wter. Even in the resting nimls the rtes were very irregulr nd for this reson the rtes were usully recorded for periods of t lest 30 min. nd the results expressed s n verge over this period. The grphs in Fig. 2 show the results of typicl experiments. Fig. 2 A shows the effect of protrcted confinement under wter. Brnchil nd eril respirtory rtes were both low before confinement. A considerble increse in ctivity ws seen immeditely following confinement s the nimls struggled to rech the surfce, nd relible estimtes of the resting brnchil rte could not be mde. After 30-60 min., however, the nimls' ctivity decresed nd long periods were spent t rest t the bottom of the chmber. During the rest of the submergence the nimls were quiet but initilly showed mrked increse in the rte of brnchil irrigtory movements. This increse continued for the first 3-5 hr. of confinement nd the brnchil respirtory rte reched levels of 25-30 bets/min. This high level ws mintined throughout the rest of the submergence. Both pressure recordings from the buccl nd operculr cvities nd direct observtion of the confined nimls indicted tht the mplitude of the brnchil movements incresed together with the frequency of respirtion. The increse in mplitude of the recorded pressure wveforms ws not constnt but decresed lmost to the resting level t times. As no chnge in the observed mplitude of the brething movements ws seen t this time the decrese in the pressures recorded ws pprently due either to chnge of gill resistnce or filure of the mouth or operculr flps to close t the correct phse of the cycle. Adult nimls hve been kept submerged for periods of up to 24 hr. With the exception of one niml (in poor generl helth), which died fter

Gseous exchnge efficiency in Protopterus ethiopicus 5 only 7 hr., ll the nimls survived the submergence without hrm. It ws noticeble, however, tht fter submergence for over 15-20 hr. the nimls' respirtory movements becme very forceful nd irregulr, nd they begn to show signs of loss of equilibrium. At the onset of the ltter symptoms the nimls were llowed ccess to ir. Air/wter Confined in moist ir Air/wter B 30 Q» 60 20 Operculr Lung 40 10 20 ]T Operculr ventiltio 30 20 - ~Air/ wter ( 3 2 4 Confined under wter 6 1 _ Air/" wter - 40 _c i 0 **> (4 60.1 I! DO 10-0 / / l 1 l 1 4 Hours 1 o X 1 10 11-20 Fig. 2. The effect of confinement in ir (upper grph) nd wter (lower grph) on the qutic nd eril respirtory frequencies. Dotted line, verge rte of lung ventiltion (breths per hour) determined for the whole experimentl period. Solid line, closed circles represent verge brnchil (operculr) respirtory rte (bets/min.) during period of 1-3 min. or recording. Immeditely following the first lung ventiltion the rte of brnchil irrigtory movements fell rpidly, usully reching the pre-submergence level in 30-60 min. The rte of lung ventiltion following the submergence period ws considerbly greter thn hd been seen before confinement, nd this rte decresed more slowly to the resting level t rte dependent on the severity of the previous confinement. All the experiments quoted bove were crried out on dult (over 200 g.) nimls. Tble 1 shows the results of preliminry experiments on smller nimls. Lrvl Protopterus (3-4 weeks old) with functionl externl gills were not ffected by confinement under well-erted wter even for periods in excess of 14 dys. No brnchil respirtory movements were seen in these nimls before or during the submergence.

6 B. R. MCMAHON In juvenile nimls the brnchil hyperirrigtion response develops grdully, with incresing size. As the nimls' size increses, however, the length of time the niml cn survive without lung ventiltion decreses. Tble i. Vible submergence time for different ges of Protopterus Averge brnchil respirtory rte Stge Lrvl Young juvenile Juveniles Adults Weight (g.) o-i i-7 SO 500 + Mximum vible submergence time At lest 15 dys At lest 6 dys One to 4 dys Up to 1 dy Norml Enforced submergence 0 0 1-5 Up to 10 1 Up to 24 1 Up to 24 Development Ext. gills functionl Mouth open, lungs functionl? Ext. gills very much reduced Int. gills functionl Both functionl Both functionl In wter L.V. L.V. In ir 4. L.V. L.V. L.V. Fig. 3. Pressures recorded from the buccl cvity before nd during exposure to ir. Upper trce: before exposure to ir. Brnchil respirtory movements seen. Lower trce: fter 1 hr. exposure to ir. Note the suppression of ll brnchil movement, even tht which normlly concludes the ir-brething cycle. L.V., Air-brething cycle (lung ventiltion). In ll experiments where the nimls were confined in moist ir the brnchil respirtory rte ws lwys low even in the pre-confinement period. Brnchil respirtory movements were usully only recorded when the nimls rose to the surfce to brethe ir, or when they were otherwise ctive. One brnchil movement hs been shown to be n integrl prt of the ir-brething cycle (McMhon, 1969), but fter 1 hr. exposure to ir no brnchil irrigtory movements re seen, even this obligtory flushing stge of the ir-brething cycle hving been suppressed (Fig. 3). The nimls were usully restless when first exposed to ir nd no mesurements of the brething rtes were mde in the first hour of ech exposure. The nimls' ctivity decresed fter this time nd recordings showed tht the lung ventiltion rte hd been mrkedly incresed (verge of ten experiments, 6 x ) despite the bundnce of ir. This incresed

Gseous exchnge efficiency in Protopterus ethiopicus 7 rte ws mintined throughout the ir exposure, which ws limited to 5 hr. or less, s it is well known tht Protopterus cn survive long periods of ir exposure s long s it is kept moist. On termintion of the ir exposure drmtic but trnsitory increse in the brnchil respirtory rte ws observed s soon s the niml ws ble to submerge the mouth (Fig. 2B). Evidence from the mesurement of pulmonry gs concentrtion Smples of pulmonry gs were removed from the lungs t intervls throughout number of nturl nd rtificilly prolonged submergences. Immeditely following lung ventiltion the oxygen concentrtion in the lung gs ws high, generlly over l 5% (> 110mm. Hg P Ot ), nd the crbon dioxide concentrtion ws low, rrely 20 fa breths»» \ Voluntry * O 1 Prolonged CO, J submergence b CO[ CO,[ submergence b 15 O U.,10 2 I 0 25 50 75 100 125 1SQ Time (min.) Fig. 4. Oxygen nd crbon dioxide concentrtion in smples of pulmonry gs withdrwn from the lung of Protopterus during prolonged submergences. Air breths re indicted by rrows. The dotted rrow indictes the ir breth following long voluntry submergence. The open circles nd squres indicte the concentrtions in smples tken during nd just fter this voluntry submergence. The closed circles nd squres indicte smples tken during submergence which ws rtificilly prolonged by preventing the niml from reching the surfce. The lines (dotted, CO] concentrtion; solid, O» concentrtion) re drwn through the points from the rtificilly prolonged submergence. more thn i-5-2-5% (11-22 mm. Hg J co,)- During the length of n verge submergence (20-25 min., t rest in the home qurium) the oxygen concentrtion fell rpidly (Fig. 4) to 4-5% (30 mm. HgP 02 ). If the submergence period ws prolonged by denying the niml ccess to the surfce the oxygen concentrtion continued to decrese, but much more slowly, reching level of O'3-O'5% (3 mm. Hg P Ot fter 150 min. The crbon dioxide concentrtion rose rpidly in the first 5-10 min. fter lung ventiltion, reching level of 4-5 % (25-30 mm. Hg P C o t )- Little or no further

8 B. R. MCMAHON increse occurred, however, nd pulmonry crbon dioxide concentrtion exceeded 5 % in only one niml fter 150 min. submergence (Fig. 4). The originl level of both gses ws lwys restored t the next lung ventiltion fter nturl submergence, but second ventiltion ws often needed to restore the levels fully when the submergence hd been prolonged for more thn one hour. Evidence from the mesurement of gs tensions in inhlnt nd exhlnt brnchil wter Smples were removed from the buccl nd operculr cvities by mens of implnted cnnule. The wter in the buccl cvity (JP/,O,) vried little from tht in the mbient wter, but chnges in the concentrtion of both oxygen nd crbon dioxide 80 r 60 - I 40 o" - 20-8 V D D t 1 I 1 2 3 Log BRR - 15 _ - 5 8 10 I 8 Fig. 5. Oxygen uptke nd crbon dioxide production in wter pssing over the gills plotted s function of the brnchil respirtory rte (BRR). PI,O, PI,O, (tension of oxygen in inhlnt wter tension of oxygen in the exhlnt wter) = oxygen uptke. -Pjt.o, -P/.o (tension of COj in the exhlnt wter tension of CO, in the inhlnt wter) = crbon dioxide production. Solid circles = P o,- Open squres = P C o,- occurred in pssge over the gills nd were detected in the exhlnt brnchil wter (P ) o 2 ). These chnges re plotted s function of respirtory rte in Fig. 5. It cn be seen tht both oxygen nd crbon dioxide re exchnged t this site, nd tht the rte of this exchnge vries with the rte of brnchil respirtion. Unfortuntely, no estimte of ventiltion volume could be mde. Evidence from the respirometry experiments The ctul consumption of oxygen nd production of crbon dioxide were mesured in the specilly designed respirometer illustrted in Fig. 1. Becuse of the difference in size of the experimentl nimls (150-600 g.), nd perhps becuse of differences in

Gseous exchnge efficiency in Protopterus ethiopicus 9 physiologicl stte, some vrition of the individul rtes of gs exchnge ws seen. Men figures for oxygen nd crbon dioxide exchnge hve been clculted from the dt obtined from nine experiments on four different nimls. These figures re presented in histogrm form in Fig. 6. 7 # Air/wter No ir Air/wter CO 2 u ml. 1 80-60- 40-. 80- u 7 60- oi E 40-20- Air/wter No ir Air/wter Fig. 6. Averge oxygen consumption nd crbon dioxide production occurring vi eril nd qutic routes in norml nimls, nimls confined under wter, nd nimls recovering from confinement under wter. Exchnge rtes re expressed in cubic millimetres per kilogrm body weight per hour (ml./kg.'vhr." 1 ). The figures re verged from number of experiments. The rnge of vrition is given in the text. The upper line indictes the totl exchnge under the stipulted conditions. The blck re indictes the proportion of totl exchnge vi the qutic route, nd the cler re the proportion pssing vi the eril route., Exchnge t gills;, exchnge in lungs. The first column shows the results obtined from nimls free to brethe ir or wter. The verge figure for totl oxygen consumption ws 62-5 c.c. oxygen/kg./hr. (rnge of vrition 27-8-86-5 ml./kg.~ 1 /hr.~ 1 ). The verge figure for crbon dioxide production ws 47*4 c.c. crbon dioxide/kg./hr. (rnge of vrition = 19-9 56-3 ml./ kg.~ 1 /hr.~ 1 CO 2 ). The clculted totl RQ for the verged results is 0-755, resonble figure for crnivorous niml. The results show tht the exchnge rtio ws very different t the two respirtory rtes. In every experiment the oxygen consumption over the pulmonry surfce ws much greter thn over the gills. Averge eril oxygen consumption ws 91-7% of the totl (rnge 86-5-94-0%). It is evident tht under these conditions the lung is the principl site for oxygen exchnge. Exmintion of the figures, however, showed tht only 32-5 % of the crbon dioxide is excreted vi the eril route, the reminder pssing vi the qutic route. In five experiments the nimls were denied ccess to the surfce for periods of

io B. R. MCMAHON bout 60 min. during the course of the experiment. Brnchil respirtory rte ws incresed by up to four times nd incresed gseous exchnge ws seen cross the gill surfce (Fig. 6, second column). Crbon dioxide production vi the gills ws incresed so tht up to 100% (verge 85 %) of the pre-confinement production now pssed by this route. The qutic oxygen consumption, however, though considerbly incresed, could provide only 17% (11 8-30*0%) of the nimls' totl pre-confinement oxygen requirements. Rtes of gs exchnge were lso mesured for 1 hr. fter the end of the confinement. A very mrked increse in the totl oxygen consumption ws seen (49*8-72*6 % bove the pre-confinement level.) The rte of lung ventiltion lso incresed nd ll the dditionl oxygen ws consumed by this route. The incresed lung ventiltion lso ffected the crbon dioxide production rtio, s on the verge 57 % of the totl crbon dioxide produced ws now eliminted vi the lungs, while the mount pssing vi the gills ws correspondingly reduced. Before evluting the results of these experiments it must be mentioned tht the method of estimtion of crbon dioxide concentrtion in the wter smples ws ccurte to 5 % only. This level of inccurcy, while high, ws not sufficient to influence the conclusions drwn from these results. DISCUSSION The experimentl evidence presented here demonstrtes clerly tht gseous exchnge t the gill surfce ccounts for very little of the totl oxygen uptke in the dult Protopterus. This is in greement with the work of Lenfnt & Johnsen (1968). The rte of brnchil respirtion is very low in nimls t rest nd the percentge utiliztion{p /Ol P EfOl )/P />Ol } xioo is very low when compred with the figures published for other fishes in Tble 2. Percentge utiliztion seen in Neocertodus Tble 2. Percentge utiliztion of oxygen t the gills of vrious fishes Dogfish Trigger fish Crp Trout Eel Neocertodus Lepidosiren Protopterus Protopterus BRR/min. 42 40-60 - 17-19 3 vr. o-5 10 Ventil. vol. /min. 248 ±42 200-330 300 133 64 315 Pi,o 2 155 151 Air st. Air st. 131 140 140 Percentge utiliztion 48 58-81 49 up to 80% 68 36-3 0-40 46 13 Source Piiper & Schumnn (1967) Hughes, G. M. (1967) Sunders, R. L. (1962) Vn Dm (1938) Vn Dm (1938) Lenfnt et l. (1966) Johnsen & Lenfnt (1967) Present survey (1969) Present survey (1969) compres with tht seen in the other fishes but tht of Protopterus is only 50 % efficient t very low irrigtion rtes. If the niml increses the rte of brnchil irrigtion, s seen in response to protrcted submersion, the percentge utiliztion flls to very low level. Oxygen consumption thus flls by over 80% when dult Protopterus re confined underwter for protrcted periods. The oxygen consumption lso flls in Neocertodus similrly confined but by only 20-25% (Grigg, 1965 c). Direct evidence s to the inefficiency of the gills in oxygen uptke is given by the respirometry experiments, where increses of brnchil respirtory rte of up to four times in confined

Gseous exchnge efficiency in Protopterus ethiopicus 11 nimls could provide only 10-30% of the nimls' oxygen requirements. The nimls were forced into oxygen debt when prevented from brething ir, nd this oxygen debt ws pid off by the mrked hyperventiltion seen once ccess to ir ws possible. The increse in qutic oxygen uptke during long submergence is, however, of vlue in llowing even dult nimls to remin submerged for reltively long periods. Crbon dioxide excretion occurs over both lung nd gill surfces, with perhps the mjor prt pssing quticlly in the resting niml. The percentge of crbon dioxide pssing vi either route cn be incresed by hyperventiltion, but, wheres modertely incresed brnchil respirtion ws ble to remove ll the crbon dioxide during qutic confinement, very mrked increse in eril respirtion ws needed to remove the crbon dioxide ccumulted during exposure to ir. If the dt obtined from direct mesurement of the respirtory medi re plotted in the form of O /CO digrms, s first used by Willmer (1934) nd more recently by Rhn & Fenn (1955), confirmtion of the results expressed bove cn be seen. Figures 7A, B show O 2 /CO 2 plots for pulmonry ir nd expired wter respectively. In Fig. 7B regression line hs been clculted from the plotted dt to show the reltionship between qutic oxygen nd crbon dioxide exchnge. Theoreticl gs exchnge lines where R = unity re lso drwn for eril nd qutic routes. The slope of the plotted regression line (R = 4*9) is considerbly greter thn R = 1 for wter, nd this figure is in good greement with the exchnge rtio of 5-4 clculted from the respirometry dt. A high qutic gs exchnge rtio indictes tht much more crbon dioxide thn oxygen is being exchnged cross the gill surfce. This imblnce could be explined by prior oxygention of the blood reducing the mount of possible oxygen uptke, or could be due to thickening of the gill epithelium such s is seen in Lepidosiren (Fullrton, 1931) which would reduce the possible exchnge for the less soluble oxygen while hving much less effect on crbon dioxide. As very little hs yet been published on the degree of seprtion occurring in the prtilly divided hert of Protopterus, the extent of the former is difficult to estimte. The lengthening of the diffusion pth would be of dptive benefit in n niml where the blood pssing through the gills my hve higher P Ot thn tht of the mbient wter. Under these conditions short diffusion pth would result in the loss of oxygen to the mbient wter nd consequent loss of efficiency of the lung. When the pulmonry gs dt re plotted on n O 2 /CO 2 digrm ll the points re seen to lie beneth the R = 1 line for ir, indicting tht more oxygen uptke thn crbon dioxide elimintion tkes plce in the lung. This conclusion is in greement with the exchnge rtio deduced from respirometry dt (R = 0-27). Above crbon dioxide tensions of 20-25 mm. Hg the slope of the reltionship pproximtes to zero, indicting tht bove this level no further crbon dioxide is eliminted into the lung, though oxygen is still being removed. The time course for gs exchnge in the lung (Fig. 4) shows tht this does, in fct, occur. This indictes tht the level of crbon dioxide in the lung is very low immeditely following lung ventiltion, but tht the tension rises very quickly s crbon dioxide diffuses into the lung from the pulmonry blood strem. Equilibrtion quickly occurs between the blood nd gs nd they remin in equilibrium while ny further crbon dioxide produced by the niml is eliminted through the gills. This explntion presupposes very much higher tension of crbon dioxide in the circulting blood thn hs been shown to occur in the blood

12 B. R. MCMAHON of the fishes studied to dte (3*3 mm. Hg P COi! in dogfish venous blood (Piiper & Schumnn, 1967); 5-7 in trout ventrl ortic blood (Stevens & Rndll, 1967); 7-7 mm. Hg in the venous blood of Neocertodus (Lenfnt et l. 1966). A high level of circulting crbon dioxide might, however, be expected in the blood of n obligtory irbrething form. In fct, Lenfnt & Johnsen (1968) show levels of up to 30 mm. Hg P c0, in the dorsl rteril blood of this niml. 60 Lung ir R=l(ir) 40 o u 20 R=l(wter) 40 80 120 60 Expired wter \R=l(ir) Nv 6 40 - \ 20 R=l (wter) _ 1 1 1 1 1 1 1 20 40 60 80 100 120 140 Fig. 7.0 2 /CO 2 digrms plotted for expired brnchil wter nd pulmonry gs in Protopterus. The lower figure shows the tensions in expired brnchil wter. The regression line ws clculted by the method of lest squres. The upper grph contins evidence from three seprte experiments, ech plotted with different symbols. Theoreticl R = i lines re shown for both ir nd wter for comprison. (R = gs exchnge rtio). The occurrence of high level of crbon dioxide in the blood of Protopterus is of considerble interest. Rhn (1966) considers tht the lungs of the emergent tetrpods, though efficient in oxygen exchnge, were much less efficient crbon dioxide exchnge mechnisms thn were the gills of the ncestrl qutic forms. This, in fct, is the cse in Protopterus (B. R. McMhon, in preprtion). Rhn postultes tht cutneous crbon dioxide exchnge route ws needed in the first terrestril nimls to complement the lung exchnge nd thus prevent dngerously incresed crbon dioxide levels in the blood. He considers tht the next step would hve been the evolution of tolernce of high crbon dioxide which would hve rendered the lung tidl ventiltion mechnism sufficient for ll gs exchnge, nd would llow the cutneous exchnger with its dditionl problems of wter loss to be bndoned.

Gseous exchnge efficiency in Protopterus ethiopicus 13 The presence of high circulting crbon dioxide levels in the qutic Protopterus, however, indictes tht the erly ir-brething fishes, including the Rhipidisti, my hve lredy evolved degree of tolernce of high crbon dioxide levels, prtly due to the development of n eril respirtory mechnism but mostly in response to the presence of high crbon dioxide levels frequently found in the environment. In this cse the ncestrl tetrpods my well hve been pre-dpted to the terrestril hbitt in this respect nd the evolution of n intermedite cutneous crbon dioxide exchnger my not hve been essentil. No mesurement hs been mde of the respirtory exchnge occurring cross the skin of Protopterus. Though the importnce of cutneous crbon dioxide exchnge hs been demonstrted in Lepidosiren (Cunninghm, 1934), the skin of the dult Protopterus is neither prticulrly thin nor prticulrly vsculr nd would not pper to be n efficient exchnge surfce. The proven efficiency of the fish gill in gseous exchnge indictes tht the skin of Protopterus is unlikely to be importnt in this role in the submerged niml, though it my be importnt during estivtion when the gills re collpsed in ir (Lenfnt & Johnsen, 1968). It hs been demonstrted tht dult Protopterus ethiopicus obtin 90% of their oxygen consumption from the eril exchnge occurring in the lungs, even when the nimls re immersed in well-erted wter. If the nimls re prevented from ventilting the lungs for /ong periods, brnchil hyperirrigtion is seen. This response cnnot provide the whole of the nimls' oxygen requirement, but the dditionl oxygen consumption, though smll, is of importnce in prolonging the possible submergence time. The results obtined by Jesse et l. (1968) for either P. ethiopicus or P. dolloi (not specified) would suggest tht the gills were of greter importnce thn is indicted here. These workers used juvenile specimens, however, in which the degree of dependence on eril respirtion is less well developed (B. R. McMhon, in preprtion). Crbon dioxide excretion cn occur vi either qutic or eril routes, nd lthough the mjor prt normlly psses over the gills the frction pssing erilly cn be incresed by hyperventiltion. As the niml cn utilize eril oxygen nd is tolernt of high externl crbon dioxide concentrtions (B. R. McMhon in preprtion), it is extremely well suited to its periodiclly hypoxic nd hypercrbic environment. Protopterus is thus more similr to Lepidosiren thn to Neocertodus, both in hbitt nd in the degree of dependence on eril respirtion. If we imgine the rhipidistin fishes s hving been similrly dpted to their rther similr environment, then they were eminently pre-dpted to colonize the terrestril hbitt. SUMMARY 1. The efficiency of gs exchnge over the lung nd gill surfces of Protopterus hs been investigted. 2. Animls confined in wter or in ir showed n incresed respirtory frequency in the remining medium, indicting tht both routes were importnt in the totl gs exchnge. 3. Direct mesurement of the oxygen nd crbon dioxide tensions of pulmonry ir nd inspired nd expired brnchil wter showed gs exchnge rtios (R) of 0-2 for

14 B. R. MCMAHON the lung nd 5-0 for the gills pproximtely, demonstrting tht more oxygen ws consumed vi the lungs nd more crbon dioxide excreted vi the gills. 4. Oxygen consumption nd crbon dioxide production were mesured directly in respirometer in which respirtory ir nd wter strems could be kept seprte except during lung ventiltion. At lest 90% of the nimls' oxygen consumption occurred in the lung, while 60% of the crbon dioxide excreted pssed vi the qutic route. 5. The results re discussed with reference to the nimls' dpttion to its environment nd with reference to the evolution of the terrestril vertebrtes. I m gretly indebted to Professor G. M. Hughes, Deprtment of Zoology, University of Bristol, in whose lbortory nd under whose supervision this work ws crried out, nd to the Science Reserch Council who provided finncil support. REFERENCES BERG, T. & STEEN, J. B. (1965). Physiologicl mechnisms for eril respirtion of the Eel. Comp. Biochem. Physiol. 15, 469-84. CARTER, G. S. & BEADLE, L. C. (1930). Notes on the hbitt nd development of Lepidosiren prdox. J. Linn. Soc. (Zool.) 37, 197-203. CUNNINGHAM, J. T. (1934). Experiments on the interchnge of oxygen nd crbon dioxide between the skin of Lepidosiren nd the surrounding wter, nd the probble emission of oxygen by the mle Symbrnchus. Proc. zool. Soc. Lond. 102, 875-87. DEAN, B. (1906). Notes on the living specimens of the Austrlin lungfish Certodus forsteri in the Zoologicl Society's collection. Proc. zool. Soc. Lond. 74, 387-436. DYE, J. F. (195 I). Clcultion of the effect of temperture on ph, free CO 2, nd the three forms of lklinity. J. Am. Wt. Wks Ass. 44, 356-72. FULLARTON, M. H. (1931). Notes on the respirtion of Lepidosiren. Proc. zool. Soc. Lond. 99, 1301-6. GRIGG, C. (1965 ). Studies of the Queenslnd lungfish Neocertodus forsteri (K). I. Antomy, histology nd functioning of the lung. Aust. J. Zool. 13, 243-53. GRIGG, C. (19656). Studies of the Queenslnd lungfish Neocertodus forsteri (Krefft). II. Therml cclimtion. Aust. J. Zool. 13, 407-11. GRIGG, C. (1965 c). Studies of the Queenslnd lungfish Neocertodus forsteri (K). III. Aeril respirtion in reltion to hbits. Aust. J. Zool. 13, 413-21. HUGHES, G. M. (1967). Experiments on the respirtion of the Trigger fish (Btistes cpriscus). Experienti 23, 1077. JESSE, J., SHUB, C. & FISHMAN, A. P. (1968). Lung nd gill ventiltion of the Africn lungfish. Respir. Physiol. 3, 267-287. JOHANSEN, K. & LENFANT, C. (1967). Respirtory function in the South Americn lungfish Lepidosiren prdox (F). J. exp. Biol. 46, 205-18. JOHANSEN, K. & LENFANT, C. (1968). Respirtion in the Africn lungfish Protopterus ethiopicus. II. Control of brething. J. exp. Biol. 49, 453-68. KERR, J, G. (1897). Hbits of Lepidosiren in the dry seson. Proc. zool. Soc. Lond. 1897. KERR, J. G. (1898). Hbits nd development of Lepidosiren. Proc. zool. Soc. Lond. 1898. LENFANT, C, JOHANSEN, K. & GRIGG, C. (1966). Respirtory properties of blood nd the pttern of gs exchnge in the lungfish Neocertodus forsteri (K). Respir. Physiol. 2, 1-21. LENFANT, C. & JOHANSEN, K. (1968). Respirtion in the Africn lungfish. I. Respirtory properties of blood nd norml ptterns of brething nd gs exchnge. J. exp. Biol. 49, 437-52. LONGMAN, F. L. S. (1928). Notes on Epicertodus. Mem. Qd Mus. 1928. MCMAHON, B. R (1969). A functionl nlysis of the qutic nd eril respirtory physiology of n Africn lungfish Protopterus ethiopicus with reference to the evolution of vertebrte lung ventiltion mechnisms. J. exp. Biol. 51, 407-30. PIIPER, J. & SCHUMANN, N. (1967). Efficiency of O, exchnge in the gills of the Dogfish Scyliorhinus stellris. Respir. Physiol. 2, 135-48. RAHN, H. & FENN, W. O. (1955). A Grphicl Anlysis of Respirtory Gs Exchnge. The O 2 /CO 2 Digrm. The Americn Physiologicl Society, Wshington, D.C. RAHN, H. (1966). Aqutic gs exchnge theory. Respir. Physiol. 1, 1-12.

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