TO FREEZE OR NOT TO FREEZE:

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TO FREEZE OR NOT TO FREEZE INSECTS IN WINTER
www.carleton.ca/kbstorey
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INSECT STRATEGIES FOR WINTER SURVIVAL
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Ball galls of Eurosta soliaginis and Elliptical
galls ofEpiblema scudderiana are found on some
species of Goldenrod Solidago altissima, S.
gigantea S. canadensis
Goldenrod, Solidago.
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Eurosta solidaginis, the goldenrod gall fly
FREEZE TOLERANT
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Epiblema scudderiana, the goldenrod gall moth
FREEZE AVOIDING
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GOLDENROD GALL INSECTS
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THEMES in Cold Hardiness

• Cryoprotection polyols, AFPs, INPs, water
  balance
• Metabolism and enzyme regulation
• Reversible phosphorylation control, kinases
  phosphatases
• Metabolic rate depression
• Anoxia / ischemia resistance
• Antioxidant defense
• Selective gene activation
• Chaperone proteins, stress response
• Transcription factors and signal transduction
  cascades

Stress resistance, Viability, Life
extension common themes across phylogeny
RECENT HIGHLIGHTS Reversible phosphorylation
controls Ischemia resistance (HIF) Chaperone
proteins (HSPs, GRPs) ER stress - Unfolded
protein response
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Reversiblephosphorylation glycerol synthesis
in Epiblema
Cold activated Glycogen phosphorylase Cold
inactivated Glycogen synthase FBPase
(inhibit gluconeogenesis)
G6PDH Glucose-6-P Dehydrogenase
KEY to 1. NADPH carbon output for polyol
synthesis 2. NADPH production for antioxidant
defense
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Epiblema G6PDH
Acclimation temperature At 15C At
-16C Vmax 54 U/mg 49 U/mg Km G6P 9 µM 16
µM P-content Low High Kinases PKA No
effect P-tases No effect PP1
Low P
High P
Why phosphorylate? Glycogen targeting? Channel
G6P into Pentose Phosphate Pathway
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SEASONAL CHANGES IN PROTEIN KINASE A PROTEIN
PHOSPHATASE -1
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CONTROL by P/deP

• UNIVERSAL method of
• stress-responsive metabolic rate depression
• - across phylogeny
• - across cell metabolism
• Na/K-ATPase, Ca-ATPase ion pumping
• Ribosomal initiation factors
• protein synthesis

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Winter activity patterns of ion motive ATPases
Activity nmol/min/mg protein
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Eurosta Effects of endogenous protein kinases
or alkaline phosphatase
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EUROSTA HYPOXIAINDUCIBLE FACTOR 1
Ischemia resistance (HIF)

• Freezing is an ischemic stress, oxygen depleted
• Over time ATP depleted and glycolytic products
  accumulate lactate, alanine

HIF-1 mediates gene expression responses that
promote hypoxic / ischemic survival. HIF-1
alpha subunit stabilized atlow O2, dimerizes,
and activates genes Does freezing trigger
HIF-1expression in Eurosta ?
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EUROSTA hif-1? transripts
At 15C
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EUROSTA HIF-1? PROTEIN
HIF-1? protein peaks in mid-winter
HIF-1? protein rises2.3-fold with cold exposure
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Why is HIF-1? up-regulated

• Selected HIF-1 mediated effects are important
• Freezing is an ischemic stress
• Cold exposure increases GP, HK PFK - related
  to both ischemia polyol synthesis
• Raise glycolytic capacity near 0C
• Maintain ischemia resistance over winter

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SHOCK PROTEINS
Chaperone proteins

• Not just for shock anymore
• Integral part of stress response by tolerant
  organisms
• Support long term homeostasis and viability
• Very important in hypometabolic states when
  ATP is limiting

HSPs heat shock proteins -
protein chaperones GRPs glucose-regulated
proteins
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EPIBLEMA HEAT SHOCK PROTEINS Freeze avoiding
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EUROSTA HEAT SHOCK PROTEINS Freeze tolerant
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EUROSTA
Seasonal patterns
Coldacclimation
Anoxia exposure
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Glucose-regulated proteins

• Endoplasmic reticulum resident chaperones
• Highly conserved across phylogeny
• GRP78 roles - protein folding and secretion -
  anti-apoptosis - ER stress sensor -
  regulator of UPR (unfolded protein response) -
  interacts with PERK, ATF6 IRE1

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GLUCOSE-REGULATED PROTEINS IN EPIBLEMA
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Grp 78 EXPRESSION IN EUROSTA
Central role of GRP78 Unstressed cell GRP78
binds Tfs ATF6,
IRE1, PERK Stressed cell unfolded proteins
increase - GRP78
releases Tfs, triggers UPR
- Tfs activate genes, incl. more GRP78
- SURVIVAL vs APOPTOSIS
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ER stress - Unfolded protein response
ER stress signaling in Eurosta
PERK signaling - 1 of 3 UPR pathways - Halt
protein synthesis
Lin-Hui Yao
Poster 109
Control at 15C vs Frozen at -16C eIF2a,
P-eIF2a No change ATF3
Up 4-fold ATF4 No
change GADD34, 153 Up 2-fold
eIF2a - eukaryotic translation initiation factor
2a GADD - growth arrest and DNA damage-inducible
protein ATF - activating transcription factor
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INSECT COLD HARDINESS

• D. McMULLEN
• P. MORIN
• T. BILGEN
• A. MUISE
• D. JOANISSE
• C. HOLDEN

• T. CHURCHILL
• T. PFISTER
• J. ZHANG
• L-H. YAO
• T. FURUSAWA
• E. KOTANI

Funded by NSERC Canada
www.carleton.ca/kbstorey
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Cytochrome Oxidase Activity
15C 4C -4C -20C 24h
1 wk 1 wk
1 wk Hypox
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Epiblema Mitochondrial Genes
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EUROSTA HIF-1?

• Drosophila HIF-1a called Sima (HIF-1ß ARNT
  Tango)
• Eurosta has 62 identity overall

• Fly HIF-1a is 1500 amino acids vs.
  mammalian 830, C. elegans 719, vs. honeybee
  and shrimp 1000
• 3 unique substitutions in bHLH domain
  compared with Drosophila honeybee

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MODEL OF UNFOLDED PROTEIN RESPONSE
ATP depletion - ER Calcium depletion - Amino acid
deprivation - Hypoxia - Ischemia - Oxidative
stress Freezing Estivation - Hibernation
ER STRESS
PERK (kinase)
eIF2a (P)
Protein synthesis inhibition
Translationally regulated
Transcription activation
PP1
CHOP
GRP78
GADD34
VEGF
Target genes
Protein folding
Pro-apoptosis
Pro-survival
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A B C D

• Eurosta solidaginis fly, 3rd instar larva
• Eurytoma obtusiventris - triggers Eurosta to
  pupate
• Eurytoma gigantea - wasp
• Mordellistena convicta - beetle

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Gall collecting
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Winter profiles of cold hardening in 2 insects
FREEZE AVOIDING FREEZE TOLERANT