Theresa Cira1, John Aigner2, Tom Kuhar2, Rob Venette3, Bill Hutchison1

1 University of Minnesota Department of Entomology2 Virginia Tech Department of Entomology

3 USDA Forest Service Northern Research Station

• Basics of insect cold hardiness

• BMSB cold hardiness

• Future directions

• Question/comments

Outline

• Short term (acute) cold

• Long term (chronic) cold• What stage overwinters?

• Where does it overwinter?

• Does it acclimate, diapause?

• What is its overwintering strategy?

• Low temperature limit?

Insect cold hardiness

How to study cold?

• Short term (acute) cold hardiness

• Long term (chronic) cold hardiness• What stage overwinters?

• Where does it overwinter?

• Does it acclimate, diapause?

• What is its overwintering strategy?

• Low temperature limit?

Insect cold hardiness

How to study cold?

Adults

Aggregations, sheltered areas

? & no

?

BMSB stats:

?

Insect cold hardiness

Insect cold hardiness

Avoid the cold Survive freezingPrevent freezing

o Migrateo Aggregateo Seek shelter

o Supercoolo Void ice nucleatorso Accumulate

cryoprotectants(e.g. antifreeze proteins)

o Control the formation of ice in the body

Photo credits L to R: David R. Tribble, Pennsylvania Department of Conservation and Natural Resources, IronChris wikipedia.org Lee 2010, Salt 1961

Not mutually exclusive

Overwintering strategies

MeasurementsSupercooling point (SCP) = lowest temp before exothermwhich indicates freezing

Mortality =

inability to walk or right itself

Insect cold hardiness

Denlinger & Lee 2010

0

0.2

0.4

0.6

0.8

1

-23 -20 -17 -14 -11 -8 -5 -2 1 4 7 10 13 16 19 22 25

Pro

po

rtio

n

Cumulative SCP

Lee 2010, Salt 1961

Temperature °C

Insect cold hardiness

Hypothetical data

Frozen

Lee 2010, Salt 1961

Temperature °C

Insect cold hardiness

Hypothetical data

0

0.2

0.4

0.6

0.8

1

-23 -20 -17 -14 -11 -8 -5 -2 1 4 7 10 13 16 19 22 25

Pro

po

rtio

n

Proportion mortality

0

0.2

0.4

0.6

0.8

1

-23 -20 -17 -14 -11 -8 -5 -2 1 4 7 10 13 16 19 22 25

Pro

po

rtio

n

Cumulative SCP Proportion mortality

Lee 2010, Salt 1961

Temperature °C

Insect cold hardiness

Hypothetical data

Chill Intolerant

0

0.2

0.4

0.6

0.8

1

-23 -20 -17 -14 -11 -8 -5 -2 1 4 7 10 13 16 19 22 25

Pro

po

rtio

n

Cumulative SCP Proportion mortality

Lee 2010, Salt 1961

Temperature °C

Insect cold hardiness

Hypothetical data

Freeze Intolerant

0

0.2

0.4

0.6

0.8

1

-23 -20 -17 -14 -11 -8 -5 -2 1 4 7 10 13 16 19 22 25

Pro

po

rtio

n

Cumulative SCP Proportion mortality

Freeze Tolerant Lee 2010, Salt 1961

Temperature °C

Insect cold hardiness

Hypothetical data

Do BMSB die before, as, or after they freeze?

Chill intolerant

Freeze intolerant

Freeze tolerant

Supercooling point

Carillo 2004

Data logger

Syringe

Polystyrene cubeCooling at a rate of 1°C

Laptop

Copper constantan thermocouple wire

Methods

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

-25 -15 -5 5 15 25

Pro

po

rtio

n

Temperature (°C)

Predicted mortality curve

Predicted SCP curve

Actual cumulative SCP

Actual mortality

SCP: n=19 bugs Mortality: n=17 bugs/each temp (mean ± 95% confidence interval)Regression curves fitted with a Weibull distribution

BMSB dies at significantly higher temperatures than they freeze (p = 0.04)Chill Intolerant

Results

Predicted and observed BMSB: Cumulative SCP & proportion mortality

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

-25 -15 -5 5 15 25

Pro

po

rtio

n

Temperature (°C)

Predicted mortality curve

Predicted SCP curve

Actual cumulative SCP

Actual mortality

Results

-18°C

Lower Temperature at which there is 90% mortality (LT90)MN winter bugs LT90 = -18°C or ~0°F

Predicted and observed BMSB: Cumulative SCP & proportion mortality

SCP: n=19 bugs Mortality: n=17 bugs/each temp (mean ± 95% confidence interval)Regression curves fitted with a Weibull distribution

= greater than -5 - 0°F

= less than -5 - 0°F

= -5 - 0°F zone

Results

• Short term (acute) cold hardiness

• Long term (chronic) cold hardiness• What stage overwinters?

• Where does it overwinter?

• Does it acclimate, diapause?

• What is its overwintering strategy?

• Low temperature limit?

Results

Adults

Aggregations, sheltered areas

? & no

?

BMSB stats:

Chill Intolerant (dies before freezing)

Do the following effect BMSB cold hardiness?

1) Season tested2) Acclimation location3) Sex

Season: Spring = April and MaySummer = June, July, and AugustFall = September, October, NovemberWinter = December and February

Location: Blacksburg, VA Harper's Ferry, WV St. Paul, MN

Sex: Male Female

Methods

MN

VA

WV

Results

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

SCP

(°C

)

Female

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

Male

WV

VA

MN

Mean supercooling points (±SEM)of BMSB

Assumptions tested with a Levene test and Shapiro-Wilk test and transformed according to Box-CoxANOVA performed at a 0.05 level and TukeyHSD used for mean separation of all main factors

Results

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

SCP

(°C

)

Female

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

Male

WV

VA

MN

Mean supercooling points (±SEM)of BMSB

Assumptions tested with a Levene test and Shapiro-Wilk test and transformed according to Box-CoxANOVA performed at a 0.05 level and TukeyHSD used for mean separation of all main factors

Results

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

SCP

(°C

)

Female

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

Male

WV

VA

MN

Mean supercooling points (±SEM)of BMSB

a aa b cc ba

Assumptions tested with a Levene test and Shapiro-Wilk test and transformed according to Box-CoxANOVA performed at a 0.05 level and TukeyHSD used for mean separation of all main factors

Results

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

SCP

(°C

)

Female

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

Spring Summer Fall Winter

Male

WV

VA

MN

Mean supercooling points (±SEM)of BMSB

Assumptions tested with a Levene test and Shapiro-Wilk test and transformed according to Box-CoxANOVA performed at a 0.05 level and TukeyHSD used for mean separation of all main factors

a

c

b

• Short term (acute) cold hardiness

• Long term (chronic) cold hardiness• What stage overwinters?

• Where does it overwinter?

• Does it acclimate, diapause?

• What is its overwintering strategy?

• Low temperature limit?

Results

Adults

Aggregations, sheltered areas

Yes & no

Varies by location, season, sex

BMSB stats:

Chill Intolerant (dies before freezing)

That’s great, but BMSB numbers are only increasing

• Short term (acute) cold hardiness

• Long term (chronic) cold hardiness• What stage overwinters?

• Where does it overwinter?

• Does it acclimate, diapause?

• What is its overwintering strategy?

• Low temperature limit?

Results

Adults

Aggregations, sheltered areas

Yes & no

Varies by location, season, sex

BMSB stats:

Chill Intolerant (dies before freezing)

• Cold drives behavior– Study the triggers for overwintering behaviors– Study feeding at colder temperatures

• Cold can be directly lethal (but ecological relevance?)– Develop an estimate for what % of population is exposed to it

(remains outdoors in winter)– What shelters are sufficient buffers

• Sublethal effects of cold may be more important for BMSB– Investigate fitness effects of sublethal temperatures– Investigate effects of multiple stressors (e.g. insecticides, time)

• Indications of phenotypic variability in cold hardiness potential– Understand the overall variability in cold tolerance– Investigate mechanisms for variability (e.g. diet, genes)– Predictions need to account for geographic acclimation

Conclusions

Acknowledgements

Virginia TechJohn Aigner, Dr. Tom Kuhar

USDA Forest ServiceDr. Rob Venette

University of MinnesotaDr. Bob Koch, Dr. Bill Hutchison, Eric Burkness, Amy

Morey, Jaana Iverson, Sarah Holle Erica Nystrom, Lindsey Christianson, Anthony Hanson, Andrea Hefty,

Kelly Nail, Derek Rosenburger, Amanda Stevens

References

Questions?