The Midland Painted Turtle (Chrysemys Picta Rnarginata)

Respiration Physiology 124 (2000) 43-50 www.elsevier.com/locate/resphysiol

The physiology of hibernation among painted turtles: the midland painted turtle (Chrysemys picta rnarginata)

Scott A. Reese a, Carlos E. Crocker apb-l, Donald C. Jackson b, Gordon R. Ultsch

a Department of Biological Sciences. University of Alabama, Tuscaloosa, AL 35487-0344, USA "Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA Accepted 21 August 2000

Abstract

Midland pa&& turtles Irottt Ãˆ&hten were siflaaerged fit 3*C in nonnoxic and anorie water. In predive, and in turtles submerged for ,fCCh, pH, [U-1, ma+$ K+I,total Mfc total Ca, lactate, glucose, and osmolaiity wore 1were determined, and plasma [HCq] was calculated. Anoxic lactate from a predive value of 4.+i~moI/Lto a 150-day to 7.189. To buffer lactate increase, total calcium and magnesium row from 3.7 and 2.6 to 58:9 aad UÃˆ nunol/L, respectively. Plasma [HCOT] was titrated from 39.2 to 4.8 mnwl/L in an& turtles. Tartfes is nonnoxic water had only minor disturbances of their acid-base and ionic statuses, associated with a much smaller increase of lactate to 23 mmol/L; there was a marked increase in hematocrit from 29.1% to 42.1%. We suggest that it is emlogic, rather than phylogenetic, relationships that determine the responses of painted turtles to prolonged submergence associated with hibernation. 0 20ft) Elsevier Science B.V. All rights reserved.

Server&: Acid-base, metabolic acidosis; Buffer, blood; Diving, aonnoxia, hypoxia; Anoxia, &Vmg turtles, Reptiles, turtles

1. Introdnctioo four well-marked subspecies. Their approximate ranges (Conant and ColKns, 1998) are C. p. dar- Painted turtles (Chrywys @&a) range from dj.9 (southern painted we) the @uth-central the east to the west coasts of the USA, and from USA, C. p. betta (western painted turtle) in the the Gulf coast to southern Canada. There are northern plains states west of the Mississippi River, C. p. mwgima (midiand painted turtle) from Tennessee northward between the Missis- *CO?4SWpading author. Td.: + 1-205-3481827; fax: + 1- gxw g^ y^ a AspaheS^ Mourn and 205-3481786. southern Quebecand Ontario, C. B-mail address: @[email protected].~a.eda (G.R. Ultsch). into and p. picta 'Freant address: of Biology, an ~~e~(eastern phted turtle) between the Appalachians Stale University, San ~ransiseo,CA 94132, USA. and the Atlantic Ocean from Georgia to Nova

0034-5687/00/$ - see front matter 0 2000 Ehewier Science B.V. AB rights reserved. PE S0034-5687(00)00188-2 2 Materials and

2.1. Animals

Adult midland painted turtles (Chrysemys picta marginata) were cofleeted during the summer of 1998 in Shiawaasee County, Michigan under per- mit from the Michigan Department of Natural Resources. Turtles were maintained in an AAALAGapproved aquatic facility at the Uni- versity of Alabama, where they were kept m well water, provided basking platforms under full- spectrum light on a 12D-12L photoperiod, and fed catfish food pellets supplemented with whole fish.

2.2. Blood sampliug and analyses

The methodologies for simulated overwintering and for blood sampling are similar to those of Ultsch et al. (1985). Briefly, in late September, we stopped feeding the turtles and in WyOctober moved them into a cold room at 15% They were separated into two equally sized groups and placed in shallow (Ã13 cm) water withaccess to air, where they were cooled at I OC/day to 3Â¡C They were maintained at 3% for three days whereupon control (predive) blood samples were taken from five turtles. Samples taken after damping the neck, decapitation ante- rior to the clamp and of the severed head. A 1 cm hole was then trephined in die plastron S.A. Sense et al./JRepimtion Phystology 124 (2000) 43-50 ewer the heart. A faeparimzed (100,000 units/L ammonium heparin in turtle ringer's solution) tuberculin syringe was used to withdraw as tamer- obic aliquot of blood ( Ã0.4 ml), which was im- mediately analyzed for pH, POB, and PCQ (Radiometer BMS 3 MK2 Blood Micro System and PHM 73 pHplood-^gasmuitor, thennostat- waslead as meter) and converted to PCO, using the linear relationship between log PCQa and.@, Bood-gas electrodes - with known gas mixtures /5a-F gas-mixing pump); the pH electrode was calibrated with precision buffers (Radiometer).

-Fig. 2. As in Fig. 1B, for glucose concentration and hemat- e. The turtle was then inverted and elevated, and a 20-gauge needle with attached catheter tubing (PE 90) was inserted into the ventricle. Blood was collected as it drained from the catheter and centrifuged (3 min at 10,000 g) and the decanted -plasma was stored at - 80aC for later analysis. Duplicate microhematocrit tubes were filled from 4-1, ...... < . . , . . , 0 30 60 00 128 160 the catheter and hematocrit determined by cen- DAYS SUBMERGED trifugation for 4 min at 13,000 g (Adams Micro- hematocrit Jl centrifuge). The plasma was later PO, of C. marginata Fig. 1. A: plasma p. controls (predive, used for determinations of [Na+] and [K+] (Ra- open triangles and left y-axis) and submerged in norm& water (op circles and right y-axis) at 3-C. Ik K!02 flf diometer FLM 3 flame photometer), [d-1 (Ra- control (open triangles) and submerged turtles; andsubmer- diometer CMT10 chloride titrator), lactate and gence, filled circles, nonnoxic submergence, open circles. The glucose (YSI 2300 Slat-Plus Analyzer), total Mg 95% c~nfidenceinterval for the predive turtles is shown as and Ca (Perldn-Elmer 280 atomic absorption parallel lines, A dashed fine connects mean values for the sampling periods for the turtles submerged in normoxie water, specfrophotometer), and osmolality (Precision and a solid line connects mean values for the sampline periods Systems pOsmette 5004). Plasma [HCOJwas cal- for the Duties submerged in anoxic water. culated using the Henderson-Hasselbalch equa-

S.A. hseet at./Riisplratilm PhysMogy 124 (2000) 43-50

Anoxic turtles had a transient increase in PC02 of 5 &g; PCO, returned to predive levels by day 50. The PC02 of turtles in normoxic water did not increase; in fact, there was a trend toward a decrease, indicating that extrapulmonary C02 elimination was adequate throughout their-'submergence (Fig. 1B). Glucose levels in both groups were low and constant except in andc turtles that were near death (Fig. 2A). Hematocrit re- mained unchanged in anoxic turtles, but was elevated by day 25 in turtles in normoxic water (Fig. 2B), reaching 42.1% by day 150 (Table 1). The low or nil of submerged turtles was associated with an increase in lactate, which rose by day IS0 to 23 mmol in turtles in normoxic water and to 185 mmol in anoxic turtles (Fig. 3A). The WCOf] in anoxic turtles was titrated to low levels (4.84 mmol) after 150 days, but never fell below 12% of control values; [HCOJ in turtles in normoxic water also fell, but final values never dropped below 59% of predive values (Fig. 3B). The pH of anoxic turtles f4steadily during their submergence, reading a, final value of 7.189 at day 150; in turtles in nonaoadc water pH fell only to 7.868 (Fig. 3C). A pH-[HCOf] (Daven- port) diagram (Fig. 4) indicates that the initial acidosis in anoxic turtles was a combined respira- tory and metabolic acidosis, but became essen- tially a pure metabolic acidosis from day 50 onward.

normoxic water the same day. The Fig. 4. Chances in acid-base status of turtles submerged in nonnoxic (open circles) or anoxic (filled circles) water at 3OC, tion from 0.0 to 1.8 mmE&+ as depicted by a pH-MCOcJ(Ihvenpori) diagram. The open in ~nnoxicwater was always above -, trianele- is the mean for oredive animals; the duration of it dropped to ld@^g. 1A). submergence is indicated n&t to each sample mean.

the northern subspecies reach pH 7.2 after > 80 days of submergence, while the southern dorsalis ,aad ionic responses of mar- attain this pH after only 20 days of submergence y similar to those found in (Fig. 7). The differences cannot necessarily be , bell& sad picta attributed to a north-south physiologic dine. as 1982; Jackson and Ultsch, the musk turtle (Stemorherus odoratus), which , 1885, 1999), but quantita- has a fairly wide latitudinal range, showed no resemble those of northern physiologic dine between northern (Michigan) . Lactate accumulated in and southern (Alabama) populations with re- toadecreasedorabsent spect to their responses to submergence in accumulated a pro- an& water at 3Â° (Ultsch aad Cochran, 1994). ose in normoxic wa- Although these data do not definitively negate 'Wwg lactjrte load due to their Bleakney's hypothesis of a bellii and dorsal& hy- .enough Oifrom the water suffi- bad origin of marginata, they do cast doubt cient to accommodate a significant aerobiosis. upon it. One possibility for explaining our re- The accumulation of lactate in both grow suits is that nwqinata is a hybrid of picto and be& two northern forms already adapted to extended overwintering periods. Another is that marginata is not a hybrid, but was already present during the Wisconsinan glaciation, being harbored in an as yet undiscovered refugium somewhere in the southern portion of its present range. A third is that Bleakney was correct, and that marginata has developed, through natural selection, an ability to tolerate long-term hibcr- nation that is similar to that of beltii and picta; however it should be noted that musk, map, and aottabaQ turtles have not developed this ability owr a similar time period. We conclude that I while our data do not point definitively to any ID in a direction that would one evolution do fbr the observed acidosis (Jackson et not seem to s for subspeciation and suggest a need for further The rate.at which turtles accumulate lactate scrutiny into the evolutionary history of the seems to he &e major factor in determining their painted turtle complex. survival during anoxic submergence (Ultsch and Jackson, I#@. Based on Bleakney's hypothesis that margmata a hybrid of bellii and Acknowledgements dorsalis, we would the response to anoxic submergence of marginata to be intermediate be- We thank Sharmilee Bansal, Marcus Jones, tween fhat of beUS and dorsalis. However, using mere LeBerte, Bradley Marker, Minghua Nie, the rate of fall in pfi during submergence in Walter Smith, and Bail Stewart for aid in vari- anoxlc water as a basis for comparison, we find ous aspects of this study. This research was sup- that the response of marg&da is much more ported by National Science Foundation grants similar to that of northern (Wisconsin) bellii and IBN-96-03934 (to GS.Q and I3N-97-28794(to noitoem (Connecticut) pieta. U&g a fall of 0.8 DCJ) and by the Northern Prarie Wildlife Re- pH wits (to 7.2) as a basis for comparison, all search Centre (to GRU). S.A. Sense et al. /Respiration Physiology 124 (2000) 43-50

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