THE EFFECT OF GRADED HYPOXIA ON THE METABOLIC RATE AND BUCCAL ACTIVITY OF A LUNGLESS SALAMANDER (DESMOGNATHUS FUSCUS)

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1 The Journl of xperimentl iology, () Printed in Gret ritin The ompny of iologists Limited J9 78 T FFT OF G YPOXI O T MTOLI T UL TIVITY OF LUGLSS SLM (SMOGTUS FUSUS) LIZT. SFO, STP. WOO GL J. TTTSLL Kent Stte University, iology eprtment, unninghm ll, PO ox 9, Kent, O, US e-mil: shefor@kent.edu ccepted Octoer; pulished on WWW ovemer The hypothesis tht the lungless slmnder esmognthus fuscus responds ctively to hypoxi ws tested. Ptterns of uccl movements [pneic period durtion, the durtion (min h ) of uccl pumping nd uccl pumping frequency], hert rte nd metolic rte (rtes of oxygen uptke nd cron dioxide output) were determined during control period ( % oxygen), hypoxic period (,,., 8 or % oxygen) nd recovery period ( % oxygen). ypoxic slmnders mintined their rte of oxygen uptke t control levels until criticl oxygen level etween nd 8 % oxygen ws reched. The rte of cron dioxide output remined constnt cross ll oxygen levels, except for significnt increse during exposure to % oxygen. The uccl ctivity of lungless slmnders ws responsive to environmentl hypoxi, with significnt stimultion during exposure to. % nd % oxygen. uccl pumping frequency ws inhiited t Summry % oxygen. ert rte ws stimulted t ll hypoxic levels except % O. uring recovery, metolic rte nd hert rte returned to control levels within min fter ll hypoxic exposures. The durtions of pneic periods incresed significntly compred with the hypoxic vlue during recovery from exposure to %,. % nd % oxygen. Overll, the nimls responded ctively to hypoxi y incresing the durtion of uccl ctivity s oxygen levels decresed. The ility of these chnges to fcilitte oxygen uptke is not known. owever, the response of the dusky slmnder to low levels of oxygen is nlogous to the hypoxic ventiltory response oserved in lunged vertertes. Key words: hypoxi, Plethodontide, esmognthus fuscus, metolic rte, slmnder, uccl pump, hypoxic ventiltory response, pnoe. Introduction dult slmnders of the fmily Plethodontide re lungless; the only surfces ville for the exchnge of respirtory gses re the skin nd the inside surfces of the mouth nd phrynx. lthough lungs re sent, the process of gs exchnge is ided in the mouth nd phrynx y the movement of the skin on the floor of the mouth; i.e. uccophryngel (or uccl) pumping (Whitford nd utchison, 9). The decresed totl respirtory surfce re ville to Plethodontids compred with lunged slmnders nd their inility to mintin gs exchnge through incresed lung ventiltion, while dequte during normoxi, could impct negtively on their ility to tolerte hypoxi. considerle mount of work hs een performed on the properties of cutneous gs exchnge nd diffusion limittion using the lungless slmnder model (Gtz et l., 97; Piiper et l., 97; Feder et l., 988). lthough the contriution to gs exchnge y the uccl cpillries is proly smll, the percentge of totl cpillries locted in the uccl cvity is greter in plethodontids (pproximtely %) thn in lunged slmnders ( %) (zopek, 9), nd chnges in uccl pumping ptterns my e importnt during times of environmentl stress. In lunged nd lungless mphiins, uccl ctivity (mesured s the totl mount of uccl pumping per minute) hs een oserved to increse with incresing temperture (esmognthus qudrmcultus nd Trich grnulos; Whitford nd utchison, 9), during hypoxi (Xenopus levis; Feder nd Wssersug, 98) nd during hypercpni (ryptornchus llegniensis; outilier nd Toews, 98). In ll cses, rtes of uccl ctivity were mesured only s the overll rte of uccl pumping per minute. owever, uccl pumping typiclly occurs in urstlike pttern, with pneic periods interspersed etween periods of ctivity. We tested the hypothesis tht the pumping of the uccl pouch might itself e responsive to hypoxi nd constitute ventiltory response to hypoxi. Since lungless slmnders re derived from lunged ncestors (uen nd oucot, 989), they could retin this response even if its potentil to id in gs exchnge is very smll. Our study looks t the pttern of these ctive nd pneic periods, rther thn the overll verge rte of uccl pumping, to exmine chnges in pumping ptterns in response to lowered oxygen levels. We lso tested the effects of hypoxi on hert rte nd metolic rte, other potentil compenstory responses to hypoxi.

2 78.. SFO, S.. WOO G. J. TTTSLL Mterils nd methods nimls usky slmnders (esmognthus fuscus (finesque)) (men mss.9±. g, men ± S..M., =) were collected t West rnch eservoir (Portge ounty, Ohio, US), etween pril nd Octoer in 998 nd 999. The slmnders were housed in the lortory t on h: h light:drk cycle for minimum of dys nd mximum of dys efore eing studied. They were provided with diet of wingless fruit flies d liitum. different group of slmnders ws exposed to ech level of hypoxi, rther thn re-exposing the sme nimls to ll oxygen levels, which fcilitted pirwise comprisons etween normoxi nd hypoxi. This method llowed ech niml to ct s its own control nd lso voided the possile effects of multiple hypoxic exposures. The numers of slmnders () exposed to ech level of hypoxi (% oxygen) were s follows: %, =7; %, =;. %, =; 8 %, =; %, =. xperimentl protocol For hypoxi testing, individul nimls were plced in ml glss chmer inside temperture-controlled cinet (model PT-, Sle Systems, enderson, evd, US) t. ch niml ws exposed to control period of % oxygen overnight (t lest h), nd ll experiments were strted etween 9: nd : h the following morning. First, control vlues were recorded t % oxygen, followed y exposure to single hypoxic gs (,,., 8 or % oxygen) for 9 min, nd then 9 min recovery period t % oxygen. Gs compositions were controlled using gs-mixing flowmeter (model GF-/MP, meron Instruments, Port rnss, Texs, US), nd gs mixtures were humidified y pssing them through wter-filled flsk. The flow rte of gs through the chmer ws mintined t ml min using mss flow meter (model 8, Sierr Instruments, Monterey, liforni, US) nd controller (version., Sle Systems, enderson, evd, US). nimls were exposed to ech gs for pproximtely min efore metolic rte dt were collected. t ech oxygen level, oxygen uptke nd cron dioxide output were mesured using flow-through respirometry (F- oxygen nlyzer with signl conditioner, Sle Systems, enderson, evd, US; resolution of. %; LI- cron dioxide nlyzer, Li-or, Lincoln, ersk, US; resolution of. %), nd men vlues were clculted over period of h. t were collected nd nlyzed using tcn V Softwre (Sle Systems, enderson, evd, US). repeted-mesures nlysis of vrince (OV) ws used to compre control vlues with hypoxic men vlues. The respirtory quotient (Q) (the rtio of oxygen uptke to cron dioxide output), ws clculted t ech oxygen level. ecuse Qs were not normlly distriuted, logrithmic conversion ws performed efore compring vlues using repeted-mesures OV. efore recording metolic rte mesurements t ech level of oxygen (i.e. control, exposure nd recovery), we used n infrred ctivity detector (model -, Sle Systems, enderson, evd, US) under the glss respirometry chmer to mesure uccl cvity movements nd hert rte. min trce ws recorded t ech oxygen level, fter llowing min of exposure to new gs mixture. From the dt trce, the following vriles could e otined: pne durtion, respirtory urst durtion, uccl pumping frequency during ech respirtory urst nd hert rte during the pneic periods (Fig. ). n overll estimte of uccl ctivity ( in min h ) ws clculted using the respirtion dt s follows: urst =, t where urst is respirtory urst durtion (s) nd t is totl oservtion time (9 s). Mens for ech uccl vrile nd for hert rte (over the min period) were compred using repeted-mesures OV with Tukey s multiplecomprison post-hoc test. ecuse of the non-norml distriution of the pneic period durtions, we lso nlyzed these vlues visully using logsurvivorship curves. This type of nlysis is routinely performed (Tyler, 979) to ssess the distriutionl chnges in espirtory urst s min ert rte pne period uccl frequency Fig.. min smple trce () of uccl ctivity nd hert rte mesured with n infrred ctivity detector from which four vriles could e determined: uccl pumping frequency within urst of ctivity, hert rte during n pneic period, respirtory urst durtion nd pneic period durtion. The trce in is s recording of the region mrked in showing how hert rte nd uccl frequency were determined. The ordinte is the intensity of the infrred signl.

3 ffects of hypoxi on the slmnder esmognthus fuscus 787 ehvior tht lsts for vrious durtions (in this cse, the pneic period or reth-hold durtion). hnges in the shpes of the log-survivorship curves give n esy ssessment of chnges in the distriution of non-normlly distriuted vrile. esults ontrol (normoxic) vlues for ll vriles tested did not differ signifcntly mong tretment groups (P>. using one-wy OV). This result llows us to ssume tht ll groups hd similr responses to control conditions, even though ech group ws composed of different individul slmnders. Metolic responses to hypoxi The rte of cron dioxide output incresed significntly in response to % oxygen (P=.), ut showed no chnge t the other oxygen levels tested (Fig. ). The rte of oxygen uptke ws unchnged from the control vlue ( % oxygen) t % oxygen exposure, ut dropped significntly (P<. in ll cses) during exposure to 8 %,. %, % nd % oxygen (Fig. ). ecovery vlues for cron dioxide output nd oxygen uptke did not differ from control vlues t ny oxygen level tested. The men respirtory quotient ws clculted t ech level nd incresed significntly (P<. in ll cses) t. %, % nd % oxygen exposure (Fig. ). ert rte response to hypoxi ert rte incresed significntly during exposure to %, 8 %,. % nd % oxygen. o chnge ws oserved during exposure to % oxygen (Fig. ). ert rte returned to control levels fter ech hypoxic exposure, demonstrting no ltent post-hypoxic effects (Fig. ). uccl responses to hypoxi uccl ctivity nd uccl pumping frequency during respirtory ursts (Figs, ) did not chnge during exposure to % nd 8 % oxygen. There ws significnt increse in uccl ctivity (Fig. ) during exposure to. % (P=.8) nd % oxygen (P=.) s result of decrese in pneic period durtion (Fig. 7), ut no chnge in uccl frequency (Fig. ). xposure to % oxygen resulted in significnt (P<.) decrese in uccl pumping frequency (Fig. ). There ws no chnge in uccl ctivity (Fig. ) or in the length of pneic periods (Fig. ) compred with the normoxic controls. pneic periods were significntly longer during recovery from %,. % nd % hypoxi (P<. for ll) thn during exposure to these oxygen levels (Fig. ). The non-norml distriution nd the consistently longer pneic periods during recovery led us to construct log-survivorship plots for pneic period durtion t ech oxygen level tested (Fig. 7) using dt comined from ll nimls exposed to given oxygen level. uring exposure to %, 8 %,. % nd % hypoxi (Fig. 7 ), greter percentge of the pneic periods recorded were of shorter durtion ( leftwrd shift in the logsurvivor curve) compred with pre-exposure dt recorded t % oxygen. uring recovery from ny level of oxygen, pneic period durtions returned to control levels, lthough longer pneic periods ( rightwrd shift in the log-survivor curve) were more frequent during recovery from exposure to % nd. % oxygen thn pre-exposure. For exmple, during Fig.. te of oxygen uptke, rte of cron dioxide output nd respirtory quotient versus the oxygen level (%). sterisks (P<.) nd doule dggers (P=.) indicte significnt difference compred with control (normoxi, % O ) vlues. Vlues re mens ± S..M. for ll nimls (see Mterils nd methods for vlues of ) t the given oxygen level. te of O uptke or O output (µl O g - h - ) 7 O uptke. O output lculted Q espirtory quotient, Q

4 788.. SFO, S.. WOO G. J. TTTSLL Men hert rte (ets min - ) F 7 7 Fig.. ormoxic, hypoxic nd recovery hert rte responses to hypoxi in lungless slmnders: () % O experiment, () % O experiment, (). % O experiment, () 8 % O experiment, () % O experiment. Vlues re mens ± S..M. over minute period for individul nimls. Within ech tretment, groups with different letters re significntly different (P<.). (F) Summry of hert rte responses t ech experimentl level of oxygen; vlues re mens ± S..M. for ll nimls tested. sterisks indicte significnt difference (P<.) from the control vlue. normoxic control redings efore exposure to. % oxygen (Fig. 7),. % of the pneic periods re longer thn s. uring exposure to. % oxygen, tht vlue drops to %. uring recovery from hypoxi, pneic period durtion increses, nd.7 % of the pneic periods re longer thn s. iscussion The most importnt finding of this study is tht the uccl ctivity nd hert rtes of lungless slmnders re responsive to environmentl hypoxi. The threshold for the uccl response occurs etween 8 % nd. % oxygen. There is significnt stimultion during exposure to. % nd % oxygen nd no response, or n inhiitory response, to % oxygen. ert rte increses in response to hypoxi t ll tested O levels, except % oxygen. The uccl nd hert rte responses coincide with the metolic response to hypoxi. When encountering hypoxi, most nimls mintin their rte of oxygen uptke down to criticl oxygen level (oxyregultion), elow which the rte of oxygen uptke conforms (oxyconformtion) to the vilility of oxygen (Pörtner nd Griesher, 99). This property hs een oserved in lunged nd in nturlly or rtificilly lungless nimls (eckench, 97; Pinder, 987; Tttersll nd outilier, 999), demonstrting tht the regultion of oxygen uptke during moderte hypoxi cn e ccomplished without

5 ffects of hypoxi on the slmnder esmognthus fuscus 789 uccl ctivity (min h - ),. 8 F Fig.. Overll response of uccl ctivity (min h ) to hypoxi: () % O experiment, () % O experiment, (). % O experiment, () 8% O experiment, () % O experiment. Within ech tretment, groups with different letters re significntly different (P<.). ch point is the clculted vlue for n individul niml. (F) Summry of uccl ctivity responses t ech experimentl level of oxygen; vlues re mens ± S..M. for ll nimls tested. sterisks indicte significnt difference (P<.) from the control vlue. lungs. In our study, the criticl oxygen level for oxygen uptke occurs etween % nd 8 % oxygen. Once elow the criticl oxygen level, the lungless slmnder my still hve some ility to fcilitte oxygen uptke, s oserved y the increse in uccl ctivity during exposure to. % nd % oxygen. lthough incresed uccl ctivity t. % nd % oxygen did not llow metolic rtes to e mintined t control vlues, it my result in rte of oxygen uptke tht is higher thn would otherwise e possile. y continuously replenishing the gseous environment of the uccl cvity, diffusion cross the uccl respirtory surfce of the slmnder should e enhnced (Gtz et l., 97). The increse in cron dioxide output ut not in oxygen uptke oserved t % oxygen, together with significnt chnges in Q, suggests tht the greter uccl ctivity my result in incresed relese of cron dioxide from reltive hyperventiltion. n increse in lood flow through cutneous cpillries (through incresed cpillry recruitment nd/or lood flow rte) in response to hypoxi could lso increse cron dioxide output, ecuse loss of cron dioxide is perfusion-limited (urggren nd Molli, 98). ron dioxide output did not continue to increse during exposure to % oxygen, ut oxygen uptke decresed, nd the resulting high respirtory quotient indictes tht the cron dioxide lost through perfusion does not hve completely metolic source. possiility for incresed production of cron

6 79.. SFO, S.. WOO G. J. TTTSLL, uccl pumping frequency (z). 8 F Fig.. esponse of uccl pumping frequency (z) to hypoxi: () % O experiment, () % O experiment, (). % O experiment, () 8% O experiment, () % O experiment. Within ech tretment, groups with different letters re significntly different (P<.). Vlues re mens ± S..M. over minute period for individul nimls. (F) Summry of uccl pumping frequency responses t ech experimentl level of oxygen; vlues re mens ± S..M. for ll nimls tested. sterisks indicte significnt difference (P<.) from the control vlue. dioxide in this sitution is the titrtion of icronte s result of decrese in p cused y the formtion of lctic cid. When considering the decreses in oxygen uptke s the severity of hypoxi increses, it is difficult to differentite etween n ctive downregultion (i.e. hypometolism) nd n uncontrolled shut-down of the system due to lck of energy supplies. In nother lungless slmnder, esmognthus qudrmcultus, ooth nd Feder (99) suspected tht the reduction in oxygen uptke during hypoxi ws prtilly due to reversile metolic downregultion, hypothesis tht hs recently een gining fvor in the field (outilier et l., 997; icks nd Wng, 999). Once hypoxi ecomes severe (etween % nd % oxygen), the slmnders respond with no chnge or with decrese in respirtory ctivity nd hert rte. This my e the point t which the slmnders egin to rely on neroic metolism (Gtz nd Piiper, 979). lthough oxygen uptke egins to fll fter reching the criticl oxygen tension, plethodontids my rely on neroiosis only fter pssing lower threshold P O elow which they cnnot further decrese energetic demnd. vidence for this is seen in other functionlly lungless nimls. For exmple, ullfrogs n ctesein treted with curre rely entirely on skin rething. In wter, these frogs do not egin to ccumulte lctte until the P O of the wter is elow mmg (.7 kp), even though eroic metolic rte decreses t P O vlues elow

7 ffects of hypoxi on the slmnder esmognthus fuscus , Men pne period durtion (s) F, Fig.. esponse of pne period durtion (s) to hypoxi: () % O experiment, () % O experiment, (). % O experiment, () 8 % O experiment, () % O experiment. Within ech tretment, groups with different letters re significntly different (P<.). Vlues re mens ± S..M. over minute period for individul nimls. (F) Summry of pne period durtion responses t ech experimentl level of oxygen; vlues re mens ± S..M. for ll nimls tested. 8 mmg (. kp) (Pinder, 987). The inhiition of uccl ctivity during severe hypoxi my reflect the iphsic (excittion inhiition) response to grded hypoxi. It my lso e dptive. When the lungless slmnder is exposed to extremely low oxygen concentrtions, decresing uccl movements my slow the diffusive loss of oxygen to the environment, t lest upon initil exposure. The oserved tchycrdi in response to low oxygen levels proly reflects n increse in sympthetic tone nd could support incresed perfusion of the cutneous respirtory surfce (outilier et l., 98). ert rte did not increse with exposure to % oxygen. More severe hypoxi ( mmg;.7 kp) hs previously een oserved to decrese hert rte in the dusky slmnder (Gtz et l., 97). In extremely hypoxic conditions, sympthetic ctivity my e inhiited, or low oxygen levels my directly inhiit hert rte. uccl pumping my lso e inhiited in severe hypoxi, s seen in the decrese in uccl pumping frequency t % oxygen. In generl, the uccl response to moderte hypoxi in the dusky slmnder is similr to those of other lunged mphiins nd reptiles. The hypoxic uccl response in these lungless slmnders is ccomplished mostly y n increse in the overll mount of uccl pumping (Fig. ) nd, in some cses, y decrese in the durtion of the pneic period (Fig. 7), ut not y chnge in the ctul uccl pumping frequency. In most mphiins, the ventiltory response to

8 79.. SFO, S.. WOO G. J. TTTSLL ormoxi ypoxi ecovery Percentge of pneic periods t. 7 7 pne period durtion, t (s) Percentge of pneic periods t pne period durtion, t (s). 7 7 pne period durtion, t (s) Fig. 7. Log-survivorship plots of pne period durtions during normoxi (), hypoxi () nd recovery () in normoxi: () % O experiment, () % O experiment, (). % O experiment, () 8 % O experiment, () % O experiment. ecorded pne period durtions (t) re plotted ginst the percentge of periods whose durtion is greter thn or equl to the given durtion for ll nimls exposed to given oxygen level. The pneic period durtion needed to ccount for 9 % of the periods recorded (i.e. where 9 % of the pneic periods re t) is mrked on ech grph. leftwrd shift in the grph represents shift to shorter reth-holds, nd rightwrd shift represents greter proportion of longer reth-holds. hypoxi is chieved y reduction in the durtion of the nonventiltory periods (pnes) nd consequent overll increse in lung ventiltion (outilier, 99). This phenomenon is demonstrted in the log-survivorship plots (Fig. 7). t most levels of hypoxi, the pneic period durtions ppered to e shorter thn during normoxi, demonstrting shift to shorter reth-holds nd wy from longer pneic periods (Fig. 7). similr pttern hs een oserved in mphiins responding to hypercpni (outilier, 98). It is uncler whether the shift in uccl rething ptterns during hypoxi is chemosensory-driven reflex or whether the uccl ctivity is controlled y higher centers in the rin (Milsom et l., 997). owever, the lmost constnt frequency of individul uccl movements cross ll levels of oxygen implies tht functionl centrl rhythm genertor (see Milsom et l., 999) exists nd tht it is not modulted y peripherl chemosensory input. s result, chnges in uccl pumping ctivity cn e ccommodted only y ltertions in the durtion of rething nd pneic periods. To complicte mtters further, the uccl rething ptterns of lungless slmnders pper to e suject to multiple levels of control (e.g. olfction nd chemosensitivity; urggren nd Just, 99). In fct, we found tht direct oservtions, smll noises nd virtions could ring out increses in uccl

9 ffects of hypoxi on the slmnder esmognthus fuscus 79 pumping tht ppered to hve little to do with ventiltory requirements. In summry, the response of the dusky slmnder to decresing oxygen levels is iphsic, showing first n increse in uccl pumping, ccomplished y shortening pne durtion nd incresing uccl ctivity periods nd, therefore, incresing minutes per hour of respirtory ctivity. This is chieved y chnging the mount, ut not the frequency, of uccophryngel pumping. elow lower limit (etween % nd % oxygen), uccl ctivity is no longer incresed. These chnges in uccl ctivity my id oxygen uptke during moderte hypoxi nd my decrese metolic costs nd curtil the loss of oxygen during severe hypoxi. We cnnot ddress the effect of this iphsic response on metolic rte on the sis of our dt. Presumly, the ncestrl lunged slmnders from which plethodontids were derived hd hypoxic drive for ventiltion, s do modern mphiins (Kruhøffer et l., 987). owever, if there hd een no pressure for selection ginst uccl response, the presence of hypoxic drive in these lungless nimls could hve een retined even if the enefit were very smll nd required no current utility. n interesting ide, which remins to e tested, is tht the vrition in response etween nimls might e sign of diminished selection pressure on the hypoxic ventiltory response fter the loss of lungs. In conclusion, the uccl response to grded hypoxi is consistent with ventiltory function; i.e. hypoxic ventiltory response, which ppers to e similr to tht of lunged vertertes. This reserch ws supported y I grnt L7. Thnks to r Lowell Orr for help with niml collection nd to Shnnon ehm for lortory help. eferences eckench,. T. (97). Influence of ody size nd temperture on the criticl oxygen tension of some plethodontid slmnders. Physiol. 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