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 low-

temperature 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 (GC-MS)

• 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=0.0001

P=0.01

P=0.002P=0.002

P=0.003

Results

• Response to freezing– Ten

metabolites changed in concentration in response to freezing

P=0.015

P=0.000

P=0.000

P=0.006

P=0.000

P=0.000

P=0.001

P=0.001

P=0.004P=0.000

Results

• Response to desiccation– Total 11

metabolites changed in concentration P=0.003 P=0.000

P=0.001

P=0.004

P=0.000

P=0.001

P=0.001

P=0.004

P=0.008

P=0.004P=0.000

Results• 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

distinct treatment-dependent clustering

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 α-

ketoglutarate, 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 temperature-dependent 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.