1. 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

2. 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

3. Home Sweet Home for the Antarctic Midge

4. Antarctic Midge Habitat

5. 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

6. 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?

7. 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

8. 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

9. 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

10. 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

11. 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=0.002 P=0.003

12. Results Response to freezing –Ten metabolites changed in concentration in response to freezing P=0.015 P=0.000 P=0.006 P=0.000 P=0.001 P=0.004P=0.000

13. 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=0.004 P=0.008 P=0.004 P=0.000

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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.