The physiology of hibernation in common map turtles ž / Graptemys geographica

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1 Ž. Comprtive Biochemistry nd Physiology Prt A The physiology of hierntion in common mp turtles ž / Grptemys geogrphic Scott A. Reese, Crlos E. Crocker,,c, Mry E. Crwile, Donld C. Jckson c, Gordon R. Ultsch, Deprtment of Biologicl Sciences, Uniersity of Alm, Tuscloos, AL 35487, USA Deprtment of Biologicl Sciences, Sn Frncisco Stte Uniersity, Sn Frncisco, CA 9413, USA c Deprtment of Moleculr Phrmcology, Physiology, nd Biotechnology, Box G3B, Brown Uniersity, Proidence, RI 091, USA Received 30 April 001; received in revised form 15 June 001; ccepted 6 June 001 Astrct Mp turtles from Wisconsin were sumerged t 3C in normoxic nd noxic wter to simulte extremes of potentil respirtory microenvironments while hiernting under ice. In predive turtles, nd in turtles sumerged for up to 150 dys, plsm PO PCO ph, Cl, N, K,,, totl Mg, totl C, lctte, glucose, nd osmollity were mesured; hemtocrit nd ody mss were determined, nd plsm HCO 3 ws clculted. Turtles in noxic wter developed severe metolic cidosis, ccumulting lctte from predive vlue of 1.7 to 116 mmoll t 50 dys, ssocited with fll in ph from to To uffer lctte increse, totl clcium nd mgnesium rose from 3.5 nd.0 to 5.7 nd 7.6 mmoll, respectively. Plsm HCO 3 ws titrted from 44.7 to 4.3 mmoll in turtles in noxic wter. Turtles in normoxic wter hd only minor disturnces of their cidse sttus nd ionic sttuses; there ws mrked increse in hemtocrit from 31.1 to 51.9%. This study nd field studies suggest tht mp turtles hve n oligtory requirement for hiernculum tht provides well-oxygented wter Ž e.g. rivers nd lrge lkes rther thn smll ponds nd swmps. nd tht this requirement is mjor fctor in determining their microdistriution. 001 Elsevier Science Inc. All rights reserved. Keywords: Common mp turtles; Grptemys geogrphic; Hierntion; Acidse lnce; Ionic lnce; Overwintering 1. Introduction The ility to tolerte prolonged sumergence y n ir-rething reptile or mphiin is Corresponding uthor. Tel.: ; fx: E-mil ddress: gultsch@iology.s.u.edu Ž G.R. Ultsch.. prerequisite for underwter hierntion. All northern species of freshwter turtles studied thus fr cn tolerte months of sumergence in normoxic wter t 3C, lthough only pinted turtles Ž Chrysemys pict. hve een shown to tolerte months of sumergence in noxic wter t tht temperture ŽJckson, 000; Ultsch, 1989; Ultsch nd Jckson, Studies to dte suggest tht there re two types of physiologicl responses to $ - see front mtter 001 Elsevier Science Inc. All rights reserved. Ž. PII: S

2 33 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A prolonged cold sumergence mong northern freshwter turtles tht re in turn dependent upon whether hierntion Ž i.e. overwintering. occurs in well-oxygented wter Ž e.g. strems nd rivers. or in hypoxic or noxic hierncul Že.g. swmps or mud, respectively.. One response, typified y musk turtles Ž Sternotherus odortus., is to ecome essentilly n qutic niml in terms of gs exchnge in normoxic wter ŽUltsch, 1988; Ultsch nd Cochrn, Musk turtles ccumulte reltively little lctte during 5 months of sumergence t 3C in normoxic wter, hve slight decrese in plsm PCO, nd mintin plsm ph; ll indicting tht they re essentilly eroic throughout the winter, utilizing extrpulmonry pthwys for gs exchnge. Softshell turtles Ž Aplone spinifer. rect similrly to musk turtles when sumerged in normoxic wter ŽJckson et l., 1984; Ultsch et l., 1984 nd unpulished dt.. In contrst, neither of these species tolertes noxic sumergence well t 3C, musk turtles surviving only pproximtely 3 weeks ŽUltsch nd Cochrn, nd softshells only pproximtely weeks Ž Reese et l., unpulished dt.. A second response is typified y pinted turtles; they cn tolerte long periods of cold noxic sumergence ut cnnot remin completely eroic if the wter is normoxic. Oxygention of the wter is eneficil to n overwintering pinted turtle in tht qutic oxygen uptke enles sustntil reduction in the rte of development of n cidosis due to much smller, ut still significnt, lctte ccumultion ŽJckson et l., 000; Reese et l., 000; Ultsch nd Jckson, 198; Ultsch et l., Thus fr, there ppers to e n inverse reltionship mong turtle species in their ility to e eroic when sumerged in normoxic wter nd their ility to tolerte noxic sumergence. The inility to tolerte low mient PO during overwintering ppers to limit northern softshell nd musk turtles to hitts tht will provide hierncul with well-oxygented wter Ž e.g. strems, rivers, nd lrge lkes., while the ility to tolerte low oxygen tension possily explins why pinted turtles re uiquitous in ll permnent odies of wter within their rnge. The ility to e eroic while overwintering might seem to e due to n efficient cutneous uptke of oxygen, s oth musk nd softshell turtles hve lrge mount of exposed nd visily vsculrized skin, which is lcking in pinted tur- tles. However, common mp turtles ŽGrptemys geogrphic. re limited to the sme sorts of hitts s softshell turtles, ut they pper morphologiclly similr, nd re closely relted to, pinted turtles. Furthermore, preliminry experimentl dt Ž Ultsch nd Jckson, suggested tht mp turtle responses to simulted hierntion re much more like those of musk nd softshell turtles thn those of pinted turtles, nd field study of nturlly overwintering mp turtles in river in Vermont indicted tht the turtles were lmost entirely eroic throughout the winter in their well-oxygented hiernculum ŽCrocker et l., Here we report on studies of simulted hierntion in common mp turtles from Wisconsin nd show tht they re entirely eroic when sumerged in normoxic wter t 3C, ut fre poorly during noxic sumergence, lethl cidosis eing ttined in pproximtely 50 dys. We conclude tht n inility to tolerte prolonged noxi, nd likely significnt hypoxi, excludes mp turtles from qutic hitts tht re suject to sustntil wintertime oxygen decrement.. Mterils nd methods.1. Animls Common mp turtles Ž Grptemys geogrphic. were collected during the summers of in L Crosse County, Wisconsin under permit from the Wisconsin Deprtment of Nturl Resources. All turtles were collected, housed nd fed similrly nd used in the winter following their collection. Turtles were housed in n AAALAC ŽAssocition for Assessment nd Accredittion of Lortory Animl Cre Interntionl. -pproved qutic fcility t the University of Alm t pproximtely 0C, using well wter neutrlized with mrle chips. They were fed ctfish food pellets supplemented with whole fish. Bsking pltforms were illuminted with plnt lights on 1D1L photoperiod... Blood smpling nd nlyses The methodologies for simulting hierntion nd for smpling lood were similr to those of Ultsch et l. Ž Briefly, in erly Octoer, we cesed feeding the turtles nd then moved them

3 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A into cold room t 15C 3 dys lter. They were split into two groups nd plced in shllow wter Ž 10 cm., where they were cooled t 1Cdy to 3C, t which they were mintined for 3 dys. Control Ž predive. lood smples were tken from five turtles over ech of two study cycles. Smples were tken y crdic puncture following decpittion nd pithing. Access to the hert ws gined vi -cm hole trephined in the plstron. A heprinized Ž unitsl mmonium heprin in turtle ringer s solution. glss syringe ws used to withdrw n neroic liquot of lood Žp- prox ml., which ws immeditely nlyzed for ph, PO, nd PCO in Rdiometer BMS 3 MK Blood Micro System nd PHM 73 phlood-gs monitor thermosttted t 3C. The CO electrode output ws red s ph Ž Rdiometer PHM 40 phion meter. nd converted to PCO using the liner reltionship etween log PCO nd ph. Blood-gs electrodes were clirted with gs mixtures supplied from Wosthoff M301-F gs-mixing pump; the ph electrode ws clirted with precision uffers ŽRdiome- ter.. The turtle ws then plced plstron upwrd on n elevted stnd, nd 0-guge needle with ttched ctheter Ž PE-90. tuing ws inserted into the ventricle; lood pumped y the hert into the ctheter ws collected into microcentrifuge tues s it drined. The tues were centrifuged Ž3 min t g. nd the decnted plsm ws stored t 80C. Two microhemtocrit tues were filled from the ctheter nd hemtocrit ws determined y centrifugtion for 4 min t g. The stored plsm ws lter used for nlysis of N nd K ŽRdiometer FLM 3 flme photometer., Cl ŽRdiometer CMT10 chloride titrtor., lctte nd glucose ŽYSI 300 Stt-Plus Anlyzer., totl Mg nd C ŽPerkin-Elmer 80 tomic sorption spectrophotometer; therefore, mesurements included ll forms of these elements, not just the free ctions., nd osmollity Ž Precision Systems Osmette Plsm HCO 3 ws clculted using the HendersonHssellch eqution Ž CO 0.081, Reeves nd pk tht depended on the lood ph Ž Jckson nd Heisler, 1983.: 6.93 for control turtles nd sumerged turtles with ph 7.93 Ž the lowest ph of control turtle. nd for turtles with ph7.93. Ech group of remining turtles ws mintined sumerged y plcing plstic grte pproximtely hlfwy down into the wter column ove them. The wter in one tnk ws mintined normoxic Ž pprox. 158 mmhg. y ertors tht drew ir from outside the cold room. A second identicl tnk ws covered with plstic lid nd nitrogen ws uled through irstones ove the grte, which mintined the wter PO t 5 mmhg, usully 0 mmhg. The wter in oth tnks ws flushed occsionlly with pre-equilirted wter t 3C, without llowing the turtles to rethe. Viility ws determined periodiclly y vigorous prodding of the hed nd extremities to elicit movements. A filure to respond ws considered evidence of deth. Even though some unresponsive turtles were found to hve wek hertet upon removl, turtles in such condition did not recover upon ir ccess nd wrming nd cn e considered ecologiclly ded; thus we consider filure to respond to e resonle viility endpoint, s hs een found to e the cse in previous studies ŽUltsch, 1988; Ultsch et l., Sumerged turtles were removed for lood smpling s descried ove, except tht they were prevented from rething y clmping the neck. A 1D1L photoperiod of muted light ws mintined throughout the experiment. Smpling ws scheduled for oth groups on dys 0 Žpredive controls., 5, 15, 5, 50, 75, 104, 15 nd 150; however, no noxic turtles survived the 5075-dy intervl..3. Sttistics Sttisticl nlyses were performed with Sttistic t the 0.05 level of significnce. Comprisons were done with MANOVA, one-wy ANOVA, nd KrusklWllis test when pproprite. StudentNewmnKeuls, Bonferonni, or Tukey s HSD tests were used for multiple comprisons. Dt re given s mens.e. Dt from single turtle smpled fter 19 dys of sumergence in normoxic wter were excluded from sttisticl nlyses. 3. Results 3.1. Viility nd mss gin Two of the seven turtles in noxi until dy 50 were ded, one ws flccid ut did hve hert-

4 334 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A et nd could e smpled, nd four were wekly responsive nd with very low lood ph tht suggested they were ner deth. These oservtions nd previous dt Ž Ultsch nd Jckson, suggest tht the limit of noxi tolernce of mp turtles under these conditions is pproximtely 50 dys. In contrst, two of five turtles sumerged in normoxic wter in seprte experiment recovered fter 180 dys, nd two more recovered fter 17 dys, when llowed ccess to ir, wrmed to room temperture overnight, nd plced in tnk with shllow wter nd sking pltform. The fifth turtle ws removed, while still quite responsive, for lood smpling fter 19 dys of sumergence. For the two turtles still live in normoxic wter fter 180 dys, mss incresed in 56.3-g turtle y 3.73% Ž 0.01%dy. nd in 48.-g turtle y 3.94% Ž 0.0%dy.. All turtles gined mss, lthough rtes vried considerly; mss gin mong turtles in normoxic wter occurred t lesser rte thn mong those in noxic wter Ž. Tle Extrpulmonry gs exchnge We ssumed tht the PO of turtles sumerged in noxic wter ws zero nd tht ny greter mesured vlue ws due to smple contmintion; these turtles re herefter referred to s noxic turtles. We used the men mesured difference from zero in smples from noxic turtles to correct the PO dt for turtles smpled from normoxic wter the sme dy. The men correction rnged from 0.3 to 0.8 mmhg. The PO of turtles in normoxic wter ws lwys very low, from 0.5 to 0.6 mmhg through dy 5 nd from 1.1 to.0 mmhg from dy 50 to 150. The PCO of noxic turtles tended to fll, lthough not significntly, throughout 50 dys of sumergence, while the PCO of turtles in normoxic wter did fll significntly during the first 5 dys of sumergence to mmhg, from n initil level of mmhg, nd then remined stle Ž. through dy 150 Fig Acidse nd ionic sttuses Blood ph of noxic turtles fell rpidly nd stedily, reching t dy 50. The ph of turtles in normoxic wter remined unchnged fter 150 dys of sumergence Ž Fig. 1.. The plsm HCO 3 in noxic turtles fell to low levels Ž mmol. fter 50 dys, ut never elow 10% of control vlues; HCO in turtles in nor- 3 Tle 1 Predive vlues for ody mss, lood gsses, cidse vriles, Hct, ions, totl C nd Mg, osmollity, nd glucose compred to the vlues for the sme vriles fter 50 dys of sumergence of Grptemys geogrphic in normoxic nd in noxic wter, nd fter 150 dys of sumergence in normoxic wter, ll t 3C Predive Normoxic Anoxic Normoxic Ž n10. wter wter wter Ž 50 dys. Ž 50 dys. Ž 150 dys. Ž n6. Ž n5. Ž n5., Weight gin Ž % Ž Ž Ž. 3,, PO Ž mmhg PCO Ž mmhg ph Ž ,, HCO Ž mmoll Ž N Ž mmoll Ž K Ž mmoll Cl Ž meql Totl Mg Ž mmoll , Totl C Ž mmoll , , Lctte Ž mmoll Glucose Ž mmoll Osmollity , Ž mosmkg plsm. Hemtocrit Ž % , Note: Vlues re menss.e. Numers in prentheses re smple sizes when different from column vlues. Significntly different from predive vlues Ž ANOVA.. Significntly different from 50-dy noxic vlues Ž ANOVA..

5 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A normoxic wter. dt re given in Tle 1, long with dt for 50 dys of sumergence in normoxic wter for comprison to finl Ž 50 dy. dt for noxic turtles. Except for N, ll mesured plsm ion concentrtions chnged significntly in noxic turtles; totl C, totl Mg, nd K incresed, nd Cl decresed. Except for decrese in totl C, no significnt ionic chnges were oserved in turtles in normoxic wter. Lctte concentrtions lso chnged in noxic turtles only, where they rose drmticlly Hemtocrit, glucose, nd osmollity Hemtocrit tended to increse with time of sumergence, ut the chnge ws not significnt y the time either group hd een sumerged 50 dys; it ws significntly elevted in turtles in normoxic wter Ž s high s 66% in one niml. fter 150 dys Ž Fig. 4.. Osmollity incresed 3.4% fter 50 dys of sumergence in noxic turtles, lrgely due to lctte ccumultion, while decresing 1.6% in turtles sumerged in normoxic wter for 150 dys, presumly due to wter uptke Ž Fig. 4.. Increses in glucose concentrtion were spordic, nd minly limited to noxic turtles tht were ner deth, in which cse they often spiked to s high s 4 mmoll ŽFig. 4c.. 4. Discussion 4.1. Sumergence in normoxic wter Fig. 1. Ž. : PCO of predive Ž open tringles. nd sumerged turtles. Anoxic sumergence, filled circles; normoxic sumergence, open circles. The 95% confidence intervl for the predive turtles is shown s prllel lines. A line connects men vlues for the smpling periods. Ž. nd Ž c.: As in Ž., for ph nd HCO. 3 moxic wter lso fell, ut finl vlues never dropped elow 58% of predive vlues Ž Fig. 1c.. A phhco Ž Dvenport. digrm Ž Fig.. 3 indictes tht there ws mild respirtory lklosis comined with severe metolic cidosis in noxic turtles, while there ws slight respirtory lklosis mong turtles in normoxic wter with no metolic cidosis. Time-course chnges in plsm ions nd lctte re given in Fig. 3f. Men predive nd finl Ž50 dys sumergence in noxic wter nd 150 dys in The only remrkle physiologicl response of Grptemys geogrphic to simulted hierntion in normoxic wter during 5 months of sumergence is non-lethl wter uptke s evidenced y the weight gin mesured in the turtles. In spite of very low lood PO Ž pprox mmhg., the turtles re clerly entirely eroic, s indicted y the lck of even slight lctte ccumultion. Metolic depression is undoutedly n importnt component of this ility, ut the nimls re nevertheless not entirely torpid. They respond to touch nd cn withdrw hed nd lims quickly. Field oservtions of nturlly hiernting G. geogrphic in Vermont confirm oth the low lood PO Ž Crocker et l., 000. nd the reltive lertness nd ility to move during nturl hierntion ŽUltsch et l., 000; Crocker,

6 336 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A Fig.. Chnges in cidse sttus of turtles sumerged in normoxic Ž open circles. or noxic Ž filled circles. wter t 3C, s depicted y phhco Ž Dvenport. digrm. The durtion of sumergence is indicted next to ech smple men. 3 personl oservtions while scu-diving t mp turtle hiernculum.. Thus, the ility to utilize extrpulmonry oxygen uptke is sufficient to supply not only resting metolic rtes, ut lso to mintin either smll eroic scope for ctivity, or enough O trnsport cpcity to repy ny oxygen det ssocited with n neroic urst of ctivity. Additionl fctors permitting sufficient O uptke nd trnsport re likely to include n extreme left-shift of the O dissocition curve Ž Mginniss et l., nd n incresed red lood cell count Ž Sunders et l., 000.; this lter effect cn e seen s n increse in hemtocrit in the mp turtles Ž Tle 1.. Extrpulmonry gs exchnge ws lso sufficient for eliminting CO, not only for turtles in normoxic wter, ut lso for those in noxic wter. A metolic depression of turtles in normoxic wter would lower the rte of CO production, here pprently so much so tht even without the Fig. 3. As in Fig. 1, for N, K, Cl, totl C, totl Mg, nd lctte.

7 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A clue concerning the ultimte limiting fctor for survivl during sumergence does pper, however, t dy 150 in the form of significnt drop in osmollity, which suggests the eginning of filure of wter lnce regultion. Moreover, one turtle sumerged for 19 dys hd ph of 7.970, HCO 3 of.33, PCO of 5.79, nd PO of 0.8 mmhg, ll of which re similr to corresponding vlues for turtles sumerged for 150 dys, in spite of lctte of 0 mmoll. The mesurements on this turtle, plus the recovery of others kept sumerged in normoxic wter for similr period, suggests tht gs exchnge with the wter nd cidse lnce is not severely compromised, even fter periods tht exceed those expected to e encountered in the field. However, this turtle hd Cl of 48 mmoll, N of 85.0 mmoll, nd n osmollity of 173 mosmkg plsm, ll of which re well elow the corresponding vlues for turtles sumerged for 150 Fig. 4. As in Fig. 1, for hemtocrit, osmollity, nd glucose. pulmonry elimintion of CO, PCO fell sustntilly. The efficcy of cutneous, nd possily uccophryngel, elimintion of CO is especilly pprent in tht turtles in noxic wter did not develop trnsient respirtory cidosis due to cid-titrtion of lood icronte, s is typiclly the cse in pinted turtles ŽUltsch nd Jckson, 198; Ultsch et l., Such titrtion undoutedly occurred, ut the resultnt CO ws eliminted so rpidly tht there ws no significnt increse in plsm PCO. After 5 months of sumergence in normoxic wter, there were no physiologiclly importnt chnges in either cidse sttus Ž Fig.., or ionic chrge distriution Žsummrized s plsm ion lnce digrm, Gmlegrm in Fig. 5.. A Fig. 5. Plsm nion nd ction concentrtions, s depicted y Gmlegrm to show chrge lnce, for turtles sumerged in normoxic nd noxic wter.

8 338 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A dys nd strongly suggest dilution of the plsm, nd likely of other ody fluid comprtments, y wter influx. 4.. Sumergence in noxic wter The responses of G. geogrphic sumerged in noxic wter qulittively resemle those of other species under similr conditions ŽUltsch nd Cochrn, 1994; Jckson et l., 1984; Ultsch, 1989.; these responses re ultimtely ll relted to the rpid nd profound increse in lctte generted y neroiosis. The protons ssocited with lctte formtion titrted HCO 3 to 10% of its predive vlues y dy 50, ut the fct there ws still mesurle icronte, long with the fct tht the initil icronte should hve ll een titrted y dy 15, indictes tht icronte ws eing replced from elsewhere, the likely source eing the skeleton, in prticulr the shell, nd the ccompnying ctions eing minly clcium nd mgnesium Ž Fig. 5. ŽJckson, 000; Jckson et l., Although the turtles developed severe lctcidosis, they were le to survive pproximtely 50 dys. Two mechnisms prevented n erlier deth. The first ws the lck of respirtory component to the cidosis; in fct there ws trend in the noxic turtles towrd compenstory respirtory lklosis in spite of the lck of ventiltory regultion of PCO. More importntly, the decrese in the SID Ž strong ion difference. ws ttenuted from wht would hve occurred if the increse in lctte ws not ioniclly compensted. Stewrt Ž hs rgued tht plsm ph is dependent vrile whose vlue is determined y three independent vriles PCO, SID, nd the sum of wek cids, with the first two eing the vriles tht re primry regultors. As mentioned, without the possiility of hyperventiltion, PCO in the sumerged turtles will e set lrgely y lnce of metolic rte nd pssive diffusion processes, perhps enhnced y cutneous vsodiltion. For the turtles, the min compenstory mechnism is regultion of the SID, where lctte must e considered s strong ion. With no chnges in ions other thn lctte, SID would quickly ecome negtive nd fll s low s 70 meql y 50 dys of noxic sumergence, with n ssocited lethl ph Ž Fig. 6.. The potentil fll in SID cn e minimized y increses in the concentrtions of strong ctions or decreses in the Fig. 6. Plsm strong ion difference Ž SID. of mp turtles with ccess to ir Ždy zero; lso shown s prllel lines is the 95% confidence intervl., sumerged in noxic wter Ž open circles., nd sumerged in noxic wter Ž filled circles.. The closed tringles depict the SID tht would hve resulted from the oserved increses in plsm lctte hd no chnges in other strong ions occurred. The verticl lines represent the mgnitude of the compenstory chnges in K, C, nd Mg concen- Ž. trtions increses nd in Cl concentrtion Ž decrese.. concentrtions of strong nions, nd oth occur. The only ion tht chnges in the wrong direction is N, which decresed fter 50 dys y pproximtely 10 meql; K, totl C, nd totl Mg ll incresed, while Cl decresed, ll of which contriuted to the SID eing reduced y 50 dys to 0.5 meql, rther thn the potentil 70 meql, 6% compenstion. In ddition to the ionic chnges, considerle lctte is likely sequestered in the shell, further defending the ex- Ž. trcellulr ph Jckson, Comprtie nd ecologicl considertions Mp turtles re closely relted to pinted turtles Ž oth fmily Emydide. nd distntly relted to musk Ž fmily Kinosternide. nd softshell turtles Ž fmily Trionychide.. However, in terms of survivl when sumerged in noxic wter, nd n ility to e completely eroic when sumerged in normoxic wter, mp turtles resemle musk nd softshell turtles more thn pinted turtles. Considering the demonstrted importnce of the shell in tolerting noxic sumergence ŽJckson, 1999., the inility of mp turtles to tolerte noxi s well s pinted turtles from the sme re is surprising, s mp turtles possess n extensive shell. A comprison of prmeters fter 50 dys of noxic sumergence ppers to supply likely explntion Ž Tle.. The key is the 3%

9 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A Tle A comprison of plsm vriles of common mp turtles Ž Grptemys geogrphic. nd western pinted turtles ŽChryse- mys pict ellii., oth from Wisconsin, fter 50 dys of sumergence in noxic wter t 3C Mp turtles Western pinted turtles Control 50 dys Control 50 dys noxic noxic ph HCO Ž mmoll PCO Ž mmhg Lctte Ž mmoll N Ž mmoll K Ž mmoll Cl Ž mmoll Mg Ž mmoll C Ž mmoll Osmollity ŽmOsmkg plsm. SID Ž meql Dt for C. p. ellii for 50 dys of noxic sumergence re from Jckson et l. Ž 000., except for ph, which is n verge of 5 nd 75-dy vlues. lower rte of lctte ccumultion in the western pinted turtles, which suggests tht they re le to depress metolic rte more thn mp turtles when noxic. The lower rte of neroic lctte production cuses lesser depression of ph, ssocited with much smller decrese in the SID. Perhps ecuse other regultory systems re functioning etter in pinted turtles, they do not hve the wter influx seen in mp turtles s n increse in osmollity. Both species will die when plsm ph flls to pproximtely Žs is the cse for ll species studied under these conditions.; however, pinted turtles re le to forestll this ftl fll in ph y their presumed lower metolic rte. Why mp turtles cn remin eroic while sumerged during hierntion while pinted turtles ccumulte significnt mounts of lctte is n open question. Two possiilities re tht mp turtles lower their metolic rte during hierntion more thn pinted turtles, or tht mp turtles hve more efficcious mechnisms for extrpulmonry oxygen uptke. The lower rte of lctte ccumultion of pinted turtles in noxic wter mentioned ove does not support the former hypothesis, which suggests tht the mp turtles re etter le to utilize dissolved oxygen. However, this conclusion remins tenttive. Mp turtles provide nother exmple Žin ddition to musk nd softshell turtles. of the correltion of reltively poor ility to tolerte noxi with well-developed ility to function s essentilly n qutic niml while hiernting in normoxic wter. The ecologicl result is tht such species pper to e limited to hitts tht will relily provide hiernculum in which the wter is well-oxygented. Thus, mp nd softshell turtles, prticulrly in the northern portions of their rnges, re typiclly riverine species or found in lrge lkes, hitts tht will not ecome hypoxic or noxic during the portion of the winter when they re ice-covered. To inhit other res, such s swmps nd smll, shllow ponds, in which pinted turtles re common, would require migrting to suitle hiernculum, strtegy tht does not pper to e used. In this regrd, musk turtles re prticulrly interesting. While softshell nd mp turtles re primrily found in the sme types of hitts in the southern portion of their rnge s in the northern, common musk turtles in the south lso inhit swmps nd shllow ponds, hitts tht they pper to void ner their northern limits. How other species of northern turtles cope with potentil noxi remins n open question. Snpping turtles Ž Chelydr serpentin. rnge s fr north s pinted turtles, nd like them, re lso found in virtully ll odies of permnent wter. Therefore, it seems likely tht they re lso more tolernt of noxi thn mp nd softshell turtles, nd we hve preliminry dt on Chelydr from Michign tht shows tht snpping turtles survive noxic sumergence t 3C more thn twice s long s mp turtles, nd four nd nine times s long s musk nd softshell turtles, respectively. Physiologicl dt on other northern species re lcking. Acknowledgements We thnk Shrmilee Bnsl, Mrcus Jones, Chere LeBerte, Brdley Mrker, Wlter Smith, nd E. Ry Stewrt for their id in vrious spects of this study. We lso thnk Nel Pisley nd the Wisconsin Deprtment of Nturl Resources for their help in otining the turtles. This reserch ws supported y Ntionl Science Foundtion grnts IBN Ž to GRU. nd

10 340 ( ) S.A. Reese et l. Comprtie Biochemistry nd Physiology Prt A IBN Ž to DCJ. nd grnt from the Northern Pririe Wildlife Reserch Center of the United Sttes Geologicl Survey Ž to GRU.. Animl use ws under the guidelines nd pprovl of the University of Alm Animl Cre nd Use Committee. References Crocker, C.E., Grhm, T.E., Ultsch, G.R., Jckson, D.C., 000. Physiology of common mp turtles Ž Grptemys geogrphic. hiernting in the Lmoille River, Vermont. J. Exp. Zool. 86, Jckson, D.C., The role of the turtle shell in cidse uffering. In: Tylor, E.W., Eggington, S., Rven, J.A. Ž Eds.., Regultion of Tissue ph in Animls nd Plnts. Cmridge University Press, Cmridge, pp Jckson, D.C., 000. Living without oxygen: lessons from the freshwter turtle. Comp. Biochem. Physiol. 15A, Jckson, D.C., Heisler, N., Intrcellulr nd extrcellulr cidse nd electrolyte sttus of sumerged noxic turtles t 3C. Respir. Physiol. 53, Jckson, D.C., Herert, C.V., Ultsch, G.R., The comprtive physiology of diving in North Americn freshwter turtles. II. Plsm ion lnce during prolonged noxi. Physiol. Zool. 57, Jckson, D.C., Crocker, C.E., Ultsch, G.R., 000. Bone nd shell contriution to lctic cid uffering of sumerged turtles Chrysemys pict ellii t 3C. Am. J. Physiol. 78, R1564R1571. Mginniss, L.A., Tpper, D.S., Miller, L.S., Effect of chronic cold nd sumergence on lood oxygen trnsport in the turtle, Chrysemys pict. Respir. Physiol. 53, 159. Reese, S.A., Crocker, C.E., Jckson, D.C., Ultsch, G.R., 000. The physiology of hierntion mong pinted turtles: the midlnd pinted turtle ŽChrysemys pict mrgint.. Respir. Physiol. 14, Reeves, R.B., Temperture-induced chnges in lood cidse sttus: ph nd PCO in inry uffer. J. Appl. Physiol. 40, Sunders, D.K., Roerts, A.C., Ultsch, G.R., 000. Blood viscosity nd hemtologicl chnges during prolonged sumergence of northern nd southern musk turtles Ž Sternotherus odortus.. J. Exp. Zool. 87, Stewrt, P.A., How to Understnd AcidBse. Elsevier Press, New York. Ultsch, G.R., Blood gses, hemtocrit, plsm ion concentrtions, nd cidse sttus of musk turtles Ž Sternotherus odortus. during simulted hierntion. Physiol. Zool. 61, Ultsch, G.R., Ecology nd physiology of hierntion nd overwintering mong freshwter fishes, turtles, nd snkes. Biol. Rev. 64, Ultsch, G.R., Jckson, D.C., 198. Long-term sumergence t 3C of the turtle Chrysemys pict ellii in normoxic nd severely hypoxic wter I. Survivl, gs exchnge, nd cidse sttus. J. Exp. Biol. 96, 118. Ultsch, G.R., Cochrn, B.M., Physiology of northern nd southern musk turtles ŽSternotherus odortus. during simulted hierntion. Physiol. Zool. 67, Ultsch, G.R., Jckson, D.C., Acidse sttus nd ion lnce during simulted hierntion in freshwter turtles from the northern portions of their rnges. J. Exp. Zool. 73, Ultsch, G.R., Herert, C.V., Jckson, D.C., The comprtive physiology of diving in North Americn freshwter turtles. I. Sumergence tolernce, gs exchnge, nd cidse lnce. Physiol. Zool. 57, Ultsch, G.R., Crwile, M.E., Crocker, C.E., Jckson, D.C., The physiology of hierntion mong pinted turtles: the estern pinted turtle, Chrysemys pict pict. Physiol. Biochem. Zool. 7, Ultsch, G.R., Grhm, T.E., Crocker, C.E., 000. An ggregtion of overwintering leoprd frogs, Rn pipiens, nd common mp turtles, Grptemys geogrphic, in northern Vermont. Cn. Field-Nturlist 114,