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

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

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?

cm 50 cm 20 cm 5 cm ONDJFM Soil Temperature (°C) Average Minimum Extreme Minimum

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

Pseudacris triseriata Range: New Mexico to Canada Mass = g ; SVL = cm

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

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

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

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

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

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

Freezing Apparatus

Supercooling Point Rebound Temperature Temperature ( o C) Time T b Profile During Freezing Exposure

Freezing Survival

Crystallization Temperature ( o C) to -6-3 to -4-2 to -3-1 to -2 % Survival Chorus Frog Survival

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?

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

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

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

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

Unfrozen Frozen Summer Glucose (  mol gFW -1 ) Chorus FrogToad MuscleLiverMuscleLiver Tissue Glucose

Unfrozen Frozen Summer Phos a (  mol min -1 gFW -1 ) Total Phos (  mol min -1 gFW -1 ) MuscleLiverMuscleLiver FrogToad

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

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)

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

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

Control5 min2 hr24 hr  mol gFW Liver glucose a a a b

Control 5 min 2 hr 24 hr Muscle glucose aa b b  mol gFW -1

activetotalpercent active Percent active form Liver glycogen phosphorylase Control 5 min 2 hr 24 hr  mol min -1 gFW -1

IndependentDependent Liver glycogen synthetase Control 5 min 2 hr 24 hr nmol min -1 gFW -1

Control5 min2 hr24 hr Liver glycogen  mol gFW -1 glucosyl units

Control5 min2 hr24 hr Muscle glycogen  mol gFW -1 glucosyl units

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

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

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 ??

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

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

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

Body Mass (g) Liver Glycogen (  mol/gFW ) R 2 = 0.143, P = 0.033

Year: 2001

Results and Conclusions High mortality in and likely related to low glucose levels Low glucose levels result from low liver glycogen, not low glycogen phosphorylase activity