Metabolomics reveals unique and shared
metabolic changes in Response to Heat
Shock, Freezing, and Desiccation in the
Antarctic midges, Belgica antarctica
M. Robert Michaud, Joshua B. Benoit,
Giancarlo Lopez-Martinez, Michael A.
Elnitsky, Richard E. Lee Jr., David L. Denlinger
Introduction
• Midge, Belgica antarctica
– 2-year life cycle,
– Larvae in frozen substrate during winter, active among
pockets of primitive vegetation growing in nutrient-rich
substrate during summer
– Adult stage is short, reproduction
• Environmental stressors
– Freezing: in midge’s hibernaculum, 0 to –7ºC
– Desiccation: winter: all available water tied up as
biologically inactive ice, summer-wind and drought
– Heat: summer, >20ºC
Home
Sweet
Home
for the
Antarctic
Midge
Antarctic Midge Habitat
Introduction
• Stress tolerance in response to temperature
– Accumulation of three cryoprotective compound: erythritol,
glucose, and trehalose
– Produce heat-shock proteins to enhance both high- and lowtemperature tolerance
– The combination of low molecular weight cryoprotectants
and constitutive HSP expression presumably enables midge
to survive the rapid temperature fluctuations
• Stress tolerance in response to heat and
desiccation
– Little information available on the physiological
mechanisms used by this species to survive heat and
desiccation
Introduction
• Metabolomics
– To obtain a broad overview of changes in physiological
response to abiotic stressors
– Sampling: extracting small molecules, metabolites
– Chromatographic analysis: gas/liquid chromatography/mass
spectrometry, or by nuclear magnetic resonance
– Data analysis: through normal peak-by-peak analysis
between treatments
– Disadvantages and advantages?
Introduction
• The purpose of this article
Apply the metabolomics approach to monitor
changes in B. antarctica , energy metabolism,
amino acids, and polyols, elicited by the major
abiotic stressors, heat s, freezing, and desiccation
Sampling
• Materials and Methods
– Collected in January 2006 from islands near Palmer Station
– Stored in the laboratory at 4ºC and 100% relative humidity
in their natural substrate
• Stress exposure
– Performed using groups of 25 larvae, hold in micro- tube
– Control treatment: homogenized at 4ºC
– Heat shock treatment: submerged for 1 h in a 30ºC 50%
ethylene glycol bath, homogenized immediately
– Freezing treatment: placed for 6 h at –10ºC in a 50%
ethylene glycol, homogenized immediately
– Desiccation treatment: washed and placed for 6 d at 4ºC,
homogenized immediately
Metabolomics
• Homogenates separated by Gas
Chromatography - Mass Spectrometry (GCMS)
• Identities of separated peaks determined
(where possible)
• Peak areas converted into response ratios for
analyses of changes in metabolite levels
Statistics
• ANCOVA to determine changes in metabolite
levels relative to controls.
• Principal Components Analysis (PCA) to
determine which changes characterized which
treatment groups
• Hierarchical Clustering to measure which
physiological responses occurred with which
treatment groups
Results
• Response to heat shock
– The response ratios of a small number of metabolites
from B. antarctica larvae were significantly altered by
heat shock
– Five metabolites changed in concentration
P=0.003
P=0.002
P=0.002
P=0.0001
P=0.01
P=0.000
Results
P=0.015
• Response to
freezing
– Ten
metabolites
changed in
concentration
in response to
freezing
P=0.001
P=0.000
P=0.000
P=0.006
P=0.000
P=0.000
P=0.001
P=0.004
P=0.004
Results
P=0.001
P=0.000
P=0.004
P=0.001
P=0.008
P=0.000
• Response to
desiccation
– Total 11
metabolites
changed in
concentration
P=0.001
P=0.003
P=0.000
P=0.004
Results
distinct treatment-dependent clustering
• PCA
– Measure the degree of
separation of each
treatment group, PC1 and
PC2 38.1% and 31.7% of
total variation
– Plotting the principal
components to determine
if treatments are
physiologically distinct
from one another - YES
– Hierarchical analysis:
changes with cold and
drying most similar
hierarchical analysis
Discussion
• Metabolic response to heat shock
– B. antarctica constitutively expresses a suite of HSPs
throughout its larval life
– Four-fold increase in the concentrations of αketoglutarate, intermediate of Krebs cycle, precursor of
amino acid biosynthesis
– The increase in α-ketoglutarate suggests that the Krebs
cycle is perturbed by heat stress in the Antarctic midge
Discussion
• Metabolic response to heat shock
– B. antarctica exhibited an overall pattern of moderate
metabolite reduction in response to heat shock
– Reduced glucose levels could act to decrease flux
through glycolysis, thereby resulting in lower serine
and glycerol levels
– Such reduction of metabolites could also be the result
of rapid utilization of energy caused by a temperaturedependent increase in metabolism
Discussion
• Metabolic response to freezing
– Erythritol, glycerol, mannitol increase, contribute to
cold survival by (1) colligative anti-freeze properties
and (2) providing the protection of membranes and
proteins; Glucose and trehalose did not change
– The urea levels increased due to freezing in the midge,
an overall nitrogen cycle perturbation is likely, but
cryoprotective role also possible
– Elevation of succinic acid indicate a general inhibition
of aerobic metabolism
– Increased nonanoic acid may allow the Antarctic midge
to repel infection while in a non-motile, frozen state
Discussion
• Metabolic response to freezing
– Free amino acid, Alanine and aspartate increase,
glycine and serine decrease
– Increased alanine, likely contribute to cold survival by
providing a less toxic alternative glycolytic end-product
than lactic acid
– Decreased glycine and serine are linked in the same
biosynthetic pathway, indicates that one or more of the
enzymes involved in serine biosynthesis may be
inhibited by freezing or one of the pathways using these
amino acids as a substrate is activated
Discussion
• Metabolic response to desiccation
– Desiccation caused the accumulation of nonanoic acid,
allow the Antarctic midge to repel infection while in a
non-motile, desiccated state
– The free amino acid pool changed, likely a
consequence of perturbation of central cellular
respiration
– Glycerol and erythritol increased - to protect
membranes and proteins
– Accumulation of isocitric and succinic acid also
indicate a general inhibition of aerobic metabolism
Conclusions
• All three stresses caused serine levels to
decline, thus serine can be used as a general
stress marker in this species.
• Most metabolic responses to environmental
stressors are not general responses but are
tailored specifically to the particular
environmental stressor.