Experimental Evolution and Freezing Tolerance in Drosophila melanogaster

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Experimental Evolution and Freezing Tolerance in
Drosophila melanogaster

• Molly Burke
• EcoEvo 208

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Drosophila as a model organism

• Short generation time
• Easy manipulation in the lab
• Genome sequenced and annotated

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Proposed study

• Select for Drosophila embryos that successfully
  hatch into viable adults after being frozen and
  thawed
• After many generations, this will result in
  populations of flies that exhibit higher freezing
  tolerance

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Previous Studies

• Selection produces organisms better at tolerating
  freezing and cold temperatures
• E. coli
• Drosophila melanogaster

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E. coli adapt to Freeze/Thaw/ Growth Cycles

• Yellow line Ancestor that has been growing at 37
  C for 20,000 generations
• Green line Bacteria that have been subjected to
  freeze/thaw/ growth cycles for 1,000 generations

Sleight, S. 2005 (unpublished)
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Selection for cold-shock tolerance in D.
melanogaster

• Treatment Adult flies were subjected to a cold
  regime of -7 C for 80-120 minutes
• After 10 generations, there was an 82 cold-shock
  tolerance increase in selected lines compared to
  controls

(Chen et al 1993)
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Introduction to Cryobiology

• Hazards of freezing
• When ice forms intracellularly, it ruptures
  membranes, denatures proteins, and does
  irreversible damage to cells
• Avoiding these hazards
• Controlling the rate of cooling
• Cryoprotectants cause cells to harden like glass
  (vitrification), and no damaging ice crystals
  form

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Cryopreserving Drosophila

• Mazur et al 1993
• 5 of frozen embryos develop into adult flies
• Steponkus et al 1990
• 18 of frozen embryos develop into adult flies

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Technical Difficulties

• Age of embryos at the time of freezing
• Collect eggs 13.5-14.5 hours after they are
  laid

http//www.flybase.net/anatomy/image-browser.html
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Technical Difficulties

• Impermeable membranes
• Remove outer chorion with bleach
• Permeabilize inner vitelline membrane

Limbourg and Zalokar, 1973
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Technical Difficulties

• Chilling sensitivity
• Studies showed that chilling injury could
  be avoided by freezing eggs at a rate of about
  20,000 C
• per minute
• This can be done by freezing eggs directly
  in liquid nitrogen

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Selection for freezing tolerance
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Selection for freezing tolerance

• Only those flies that are freeze-tolerant will
  survive and contribute to the next generation
• Only very few flies will survive, so allow a few
  normal generations to take place before
  subjecting them to another treatment
• Over time, expected percentages of
  freeze-tolerant flies will increase

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Generalized Design
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Comparative Study

• Candidate gene sequencing
• Gene expression analyses
• Transcript profiling with DNA microarrays
• Component analysis
• Ice affinity purification (Graham and Davies 2005)

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References

• Sleight S. 2005. Society for the Study of
  Evolution Annual Conference (presentation of
  unpublished data)
• Chen C.-P. et al 1993. Ecological Entomology 18
  184-190
• Mazur P. et al 1993. Cryobiology 30 45-73
• Steponkus P.L. et al 1990. Nature 345 170-172
• Limbourg B. and Zalokar M. 1973. Developmental
  Biology 35 382-387
• Graham L. and Davies P. 2005. Science 310 461