To Freeze or Not to Freeze:To Freeze or Not to Freeze:Strategies and Mechanisms of Strategies and Mechanisms of

Overwintering in Terrestrial AnuransOverwintering in Terrestrial Anurans

David SwansonDept. 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?

-8

-4

0

4

8

12100 cm

50 cm

20 cm

5 cm

O N D J F M

-18

-12

-6

0

6

12

So

i l T

emp

erat

ure

(°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 CanadaMass = 0.5 - 2.0 g ; SVL = 1.9 - 3.5 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

1 Captured in Sept-Oct2 Food & water, natural photoperiod, room

temperature (23oC) until early November3 Food removed, 10oC, total darkness for 2

weeks4 Temperature reduced to 2oC in mid-

November5 Freezing expts. in Jan-Feb

Freezing Protocol

1 Placed in chamber at -1oC (dry or moist)2 Chamber temperature decreased 1oC/h3 Chamber temperature dropped until

freezing exotherm occurs (sometimes initiated by contact with external ice)

4 Chamber temperature maintained at -2.5oC to -4.5oC for 24 h

5 Thawing at 2oC and Recovery Tests

Freezing Apparatus

Supercooling Point

Rebound Temperature

Te

mp

era

ture

(oC

)1

0

-1

-2

-3

-4

Time

Tb Profile During Freezing Exposure

Species Group n SCP (C) RT (C)

Frog Dry 10 -5.11.0 -1.10.1

Toads Dry 7 -4.21.0 -0.90.2

Spadefoot Dry 9 -4.30.7 -1.50.4

Frog Wet 4 -3.60.7 -0.30.1

Toads Wet 2 -1.5 -0.5

Freezing Survival

Species n % Survival

Frog 17 47

Toads 6 0

Spadefoot 6 0

Crystallization Temperature (o C)

0

20

40

60

80

100

-4 to -6 -3 to -4 -2 to -3 -1 to -2

% S

urv

ival

4

4

5

4Chorus 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

Pi

glycogen phosphorylase (a)

phosphoglucomutase

Piglucose-6-phosphatase

Glucose F-6-P F-1, 6-P2

PFK-1

glycogen synthetase

glucose conc. in tissuesglucose 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

0

10

20

30

40

50

250300

Unfrozen

Frozen

Summer

0

4

8

12

16

Glu

cose

(m

ol

gF

W-1)

Chorus Frog Toad

Muscle Liver Muscle Liver

Tissue Glucose

0

10

20

30

40UnfrozenFrozenSummer

0

20

40

60

Ph

os

a (

mo

l min

-1 g

FW

-1)

To

tal P

ho

s (

mo

l min

-1 g

FW

-1)

Muscle Liver Muscle LiverFrog Toad

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

Control 5 min 2 hr 24 hr

mo

l g

FW

-1

0

50

100

150

200

250

300

350 Liver glucose

aa a

b

Control 5 min 2 hr 24 hr

0

5

10

15

20

25

30

35Muscle glucose

a a

b

b

mo

l g

FW

-1

0

20

40

60

80

100

0

20

40

60

80

100

active total percent active

Pe

rce

nt

acti

ve

form

Liver glycogen phosphorylase

Control5 min2 hr24 hr

mo

l m

in-1 g

FW

-1

0

100

200

300

400

500

600

700

800

900

Independent Dependent

Liver glycogen synthetase

Control5 min2 hr24 hr

nm

ol m

in-1 g

FW

-1

Control 5 min 2 hr 24 hr

0

200

400

600

800

1000

1200

1400

1600

1800 Liver glycogenm

ol g

FW

-1 g

luc

osy

l un

its

0

20

40

60

80

100

120

140

Control 5 min 2 hr 24 hr

Muscle glycogenm

ol g

FW

-1 g

luc

osy

l un

its

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

Pi

glycogen phosphorylase (a)

phosphoglucomutase

Piglucose-6-phosphatase

Glucose F-6-P F-1, 6-P2

PFK-1

glycogen synthetase

glucose conc. in tissuesglucose 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

Pi

glycogen phosphorylase (a)

phosphoglucomutase

Piglucose-6-phosphatase

Glucose F-6-P F-1, 6-P2

PFK-1

glycogen synthetase

glucose conc. in tissuesglucose conc. in tissues

no change with freezingno change with freezing

Chorus frog Liver

inhibited by freezing ??

Freezing Survival differed among Winters in Chorus Frogs

0

20

40

60

80

100

1996 1998-1999 1999-2000

% s

urv

iva

l

n = 12n = 4 n = 8

Chorus Frog Freezing Survival

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

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

1996 1998-1999 1999-2000

mea

n bo

dy m

ass

(g)

n = 4 n = 8 n = 12

0

20

40

60

80

100

1996 1998-1999 1999-2000

% s

urvi

val

n = 12n = 4 n = 8

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.

0

5

10

15

20

25

control frozen Edwards (frozen)

mu

scle

glu

cose

(m

ol/g

FW

)

b

aB

A

0

20

40

60

80

100

120

140

160

180

200

220

240

control frozen Edwards (frozen)

live

r g

luco

se (

mo

l/gF

W)

b

aB

A

0

100

200

300

400

500

600

700

800

900

control frozen Edwards (unfrozen)

live

r g

lyco

ge

n (

mo

l/gF

W)

a

aB

A

0

10

20

30

40

50

60

70

80

90

100

control frozen Edwards (frozen)

live

r %

act

ive

gly

cog

en

ph

osp

ho

ryla

se

a

aAA

Body Mass (g)

0.4 0.6 0.8 1.0 1.2 1.4

Liv

er G

lyco

gen

(

mo

l/g

FW

)

0

200

400

600

800

1000

1200 R2 = 0.143, P = 0.033

Spring Summer Fall0

10

20

30

40

50

100

200

300

400

500

600

700

800

Liver Glucose Liver Glycogen

Glu

co

se

(m

ol/g

FW

)

Gly

cog

en

(m

ol/g

FW

)

0.0

0.4

0.8

1.2

1.6

Mas

s (g

)Sp S F

Year: 2001

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