1. To Freeze or Not to Freeze: Strategies and Mechanisms of Overwintering in Terrestrial Anurans David Swanson Dept. of Biology, Univ. South Dakota

3. Anuran Overwintering Strategies Aquatic Terrestrial Burrowers Terrestrial - Shallow Freezing Tolerance vs. Freezing Avoidance

4. Freezing Tolerance in Anurans Present in species using shallow terrestrial hibernacula Absent in burrowers or aquatic overwintering species Burrowing anurans little studied - Exposed to subfreezing temps?

5. -8 -4 0 4 8 12 100 cm 50 cm 20 cm 5 cm ONDJFM -18 -12 -6 0 6 12 Soil Temperature (°C) Average Minimum Extreme Minimum

6. STUDY SPECIES Terrestrial Burrowers –Great Plains Toad, Bufo cognatus –Woodhouse’s Toad, Bufo woodhousii –Plains Spadefoot, Spea bombifrons Terrestrial - Shallow –Chorus Frog, Pseudacris triseriata

7. Pseudacris triseriata Range: New Mexico to Canada Mass = 0.5 - 2.0 g ; SVL = 1.9 - 3.5 cm

8. Spea bombifrons Range: n. Mexico to s. Canada; Mass = 2-20 g

9. Bufo cognatus Range: n. Mexico to s. Canada; Mass = 3-35 g

10. Bufo woodhousii Range: Texas to North Dakota, Montana; Mass = 2-35 g

11. Hypothesis # 1 Toads and Spadefoot will not be freezing tolerant Chorus Frog will be freezing tolerant

12. Acclimation Protocol 1Captured in Sept-Oct 2Food & water, natural photoperiod, room temperature (23 o C) until early November 3Food removed, 10 o C, total darkness for 2 weeks 4Temperature reduced to 2 o C in mid- November 5Freezing expts. in Jan-Feb

13. Freezing Protocol 1Placed in chamber at -1 o C (dry or moist) 2Chamber temperature decreased 1 o C/h 3Chamber temperature dropped until freezing exotherm occurs (sometimes initiated by contact with external ice) 4Chamber temperature maintained at -2.5 o C to -4.5 o C for 24 h 5Thawing at 2 o C and Recovery Tests

14. Freezing Apparatus

15. Supercooling Point Rebound Temperature Temperature ( o C) 1 0 -2 -3 -4 Time T b Profile During Freezing Exposure

17. Freezing Survival

18. Crystallization Temperature ( o C) 0 20 40 60 80 100 -4 to -6-3 to -4-2 to -3-1 to -2 % Survival 4 4 5 4 Chorus Frog Survival

19. Freezing Tolerance in Anurans Documented in 5 species of frogs Can tolerate up to 65-70% of total body water as ice How is this accomplished?

20. Aspects of Anuran Freezing Tolerance Low molecular weight carbohydrates (glucose and glycerol) serve anti-freeze and cryoprotectant functions Accumulation not anticipatory for those frogs using glucose Liver glycogen is the source Accumulation associated with changes in enzyme activities

21. GLYCOGEN GLUCOSE-1-P GLUCOSE-6-P GLUCOSE PiPi glycogen phosphorylase (a) phosphoglucomutase PiPi glucose-6-phosphatase GlucoseF-6-PF-1, 6-P 2 PFK-1 glycogen synthetase  glucose conc. in tissues freezing induces  g-phos activity inhibited by freezing Wood frog Liver inhibited by freezing

22. Hypothesis # 2 Chorus Frogs will mobilize glucose and show a pattern of enzyme activation during freezing similar to Wood Frogs Toads will fail to mobilize glucose and enhance enzyme activities during freezing

23. Methods Acclimation and Freezing Exposure treatments similar (–2.5°C for 24 h), except inoculated freezing; Frozen compared to unfrozen controls Leg muscle and liver were removed on ice for both frozen and control groups Assays for tissue glucose and glycogen phosphorylase activity Also measured unfrozen summer animals

24. 0 10 20 30 40 50 250 300 Unfrozen Frozen Summer 0 4 8 12 16 Glucose (  mol gFW -1 ) Chorus FrogToad MuscleLiverMuscleLiver Tissue Glucose

25. 0 10 20 30 40 Unfrozen Frozen Summer 0 20 40 60 Phos a (  mol min -1 gFW -1 ) Total Phos (  mol min -1 gFW -1 ) MuscleLiverMuscleLiver FrogToad

26. Results and Conclusions - 1 Chorus Frogs accumulate glucose, toads don’t Glucose accumulation associated with higher liver phos activity in frogs Development of freezing tolerance likely associated with winter increase in phos activity

27. Results and Conclusions - 2 Liver phosphorylase activity did not increase after freezing in chorus frogs Differs from other freeze-tolerant frogs One possible reason for this difference: transient increase in chorus frogs (increased activity for several hrs after freezing, but return to normal by 24 h in frozen state)

28. Hypothesis # 3 No transient elevation in phosphorylase activity will occur in chorus frogs Glycogen synthetase will not be inhibited by freezing

29. Methods Same acclimation and freezing methods as previously Freezing Exposure Treatments: –5 min –2 hr –24 hr Measured Tissue Glucose, Glycogen, Phosphorylase, Synthetase

30. Control5 min2 hr24 hr  mol gFW -1 0 50 100 150 200 250 300 350 Liver glucose a a a b

31. Control 5 min 2 hr 24 hr 0 5 10 15 20 25 30 35 Muscle glucose aa b b  mol gFW -1

32. 0 20 40 60 80 100 0 20 40 60 80 100 activetotalpercent active Percent active form Liver glycogen phosphorylase Control 5 min 2 hr 24 hr  mol min -1 gFW -1

33. 0 100 200 300 400 500 600 700 800 900 IndependentDependent Liver glycogen synthetase Control 5 min 2 hr 24 hr nmol min -1 gFW -1

34. Control5 min2 hr24 hr 0 200 400 600 800 1000 1200 1400 1600 1800 Liver glycogen  mol gFW -1 glucosyl units

35. 0 20 40 60 80 100 120 140 Control5 min2 hr24 hr Muscle glycogen  mol gFW -1 glucosyl units

36. Results Summary Glucose accumulated rapidly Neither phosphorylase nor synthetase activities varied with time in liver Liver glycogen did not vary with time, but muscle glycogen increased – so muscle glycogen is not the source for increasing glucose in muscle

37. GLYCOGEN GLUCOSE-1-P GLUCOSE-6-P GLUCOSE PiPi glycogen phosphorylase (a) phosphoglucomutase PiPi glucose-6-phosphatase GlucoseF-6-PF-1, 6-P 2 PFK-1 glycogen synthetase  glucose conc. in tissues freezing induces  g-phos activity inhibited by freezing Wood frog Liver inhibited by freezing

38. GLYCOGEN GLUCOSE-1-P GLUCOSE-6-P GLUCOSE PiPi glycogen phosphorylase (a) phosphoglucomutase PiPi glucose-6-phosphatase GlucoseF-6-PF-1, 6-P 2 PFK-1 glycogen synthetase  glucose conc. in tissues no change with freezing Chorus frog Liver inhibited by freezing ??

39. Freezing Survival differed among Winters in Chorus Frogs Chorus Frog Freezing Survival

40. Methods Poor survival in some years led us to question why survival differs among years 1998-99 and 1999-2000 frogs: 24-h frozen treatment –immediately euthanized –measured glucose and liver glycogen Compare values to 1996 winter data where frogs had good survival

42. Body size and freezing survival Body size (mass) and rate of cooling smaller frogs may cool faster cooling rate in 1998-1999 and 1999- 2000 was lower than in 1996 Liver glycogen reserves may be limited by body size Comparisons of 1998 and 1999 studies with 1996.

47. Body Mass (g) 0.40.60.81.01.21.4 Liver Glycogen (  mol/gFW ) 0 200 400 600 800 1000 1200 R 2 = 0.143, P = 0.033

48. Year: 2001

49. Results and Conclusions High mortality in 1998-1999 and 1999-2000 likely related to low glucose levels Low glucose levels result from low liver glycogen, not low glycogen phosphorylase activity