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Journal of Comparative Physiology B

, Volume 177, Issue 7, pp 753–763 | Cite as

Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (Sarcophaga crassipalpis): a metabolomic comparison

  • M. Robert MichaudEmail author
  • David L. Denlinger
Original Paper

Abstract

Flesh flies can enhance their cold hardiness by entering a photoperiod-induced pupal diapause or by a temperature-induced rapid cold-hardening process. To determine whether the same or different metabolites are involved in these two responses, derivatized polar extracts from flesh flies subjected to these treatments were examined using gas chromatography–mass spectrophotometry (GC–MS). This metabolomic approach demonstrated that levels of metabolites involved in glycolysis (glycerol, glucose, alanine, pyruvate) were elevated by both treatments. Metabolites elevated uniquely in response to rapid cold-hardening include glutamine, cystathionine, sorbitol, and urea while levels of β-alanine, ornithine, trehalose, and mannose levels were reduced. Rapid cold-hardening also uniquely perturbed the urea cycle. In addition to the elevated metabolites shared with rapid cold-hardening, leucine concentrations were uniquely elevated during diapause while levels of a number of other amino acids were reduced. Pools of two aerobic metabolic intermediates, fumarate and citrate, were reduced during diapause, indicating a reduction of Krebs cycle activity. Principal component analysis demonstrated that rapid cold-hardening and diapause are metabolically distinct from their untreated, non-diapausing counterparts. We discuss the possible contribution of each altered metabolite in enhancing the overall cold hardiness of the organism, as well as the efficacy of GC–MS metabolomics for investigating insect physiological systems.

Keywords

Insect Diapause Amino acid Polyol Metabolism 

Abbreviations

ANOVA

Analysis of variance

GC–MS

Gas chromatography–mass spectrometry

h

hour

NMR

Nuclear magnetic resonance

PCA

Principal component analysis

Notes

Acknowledgments

The authors of this manuscript thank Richard Sessler of the Ohio State Campus Chemical Instruments Center and Amelia Brown for their technical contributions to this manuscript. The authors also appreciate the reviewers of this article for their useful suggestions and comments. This study was funded by the National Science Foundation (#IOB-0416720) and complies with all federal, state, and local laws.

References

  1. Bino RJ, Hall RD, Fiehn O, Kopka J, Saito K, Draper J, Mikolau BJ, Mendes P, Roessner-Tunali U, Beale MH, Trethewey RN, Lange BM, Wurtele ES, Sumner LW (2004) Potential of metabolomics as a functional genomics tool. Trends Plant Sci 9:418–425PubMedCrossRefGoogle Scholar
  2. Buchholz A, Hurlebaus J, Wandrey C, Takors R (2002) Metabolomics: quantification of intracellular metabolite dynamics. Biomol Eng 19:5–15PubMedCrossRefGoogle Scholar
  3. Chen CP, Denlinger DL, Lee RE (1987) Cold-shock injury and rapid cold hardening in the flesh fly Sarcophaga crassipalpis. Physiol Zool 60:297–304Google Scholar
  4. Chen CP, Denlinger DL (1990) Activation of phosphorylase: response to cold and heat stress in the flesh fly, Sarcophaga crassipalpis. J Insect Physiol 36:549–554CrossRefGoogle Scholar
  5. Chen CP, Lee RE, Denlinger, DL (1991) Cold shock and heat shock: a comparison of the protection generated by pretreatment at less severe temperatures. Physiol Entomol 16:19–26Google Scholar
  6. Chinnasamy G, Bal AK (2003) Seasonal changes in carbohydrates of perennial root nodules of beach pea. J Plant Physiol 160:1185–1192PubMedCrossRefGoogle Scholar
  7. Chino H (1958) Cabohydrate metabolism in the diapause egg of the silkworm, Bombyx mori II. Conversion of glycogen into sorbitol and glycerol during diapause. J Insect Physiol 2:1–12CrossRefGoogle Scholar
  8. Chattopadhyay MK, Kern R, Mistou M-Y, Dandekar AM, Uratsu SL, Richarme G (2004) The chemical chaperone proline relieves the thermosensitivity of a dnaK deletion mutant at 42°C. J Bacteriol 186:8149–8152PubMedCrossRefGoogle Scholar
  9. Churchill TA, Storey KB (1989) Metabolic consequences of rapid cycles of temperature change for freeze-avoiding vs. freeze tolerant insects. J Insect Physiol 35:579–586CrossRefGoogle Scholar
  10. Churchill TA, Storey KB (1996) Organ metabolism and cryoprotectants synthesis during freezing in spring peepers Pseudacris crucifer. Copeia 1996:517–525Google Scholar
  11. Constanzo JP, Lee RE Jr (2005) Cryoprotection by urea in a terrestrially-hibernating frog. J Exp Biol 208:4079–4089CrossRefGoogle Scholar
  12. Denlinger DL (1972) Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biol Bull 142:11–24CrossRefGoogle Scholar
  13. Denlinger DL, Willis JH, Fraenkel G (1972) Rates and cycles of oxygen consumption during pupal diapause in Sarcophaga flesh flies. J Insect Physiol 18:871–882PubMedCrossRefGoogle Scholar
  14. Diamant S, Eliahu N, Rosenthal D, Goloubinoff P (2001) Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem 276:39586–39591PubMedCrossRefGoogle Scholar
  15. Dordea M, Floca L, Perseca T (1987) Variation of the content of free amino acids in eri silkworm Philosamia ricini pupae during diapause as influenced by the treatment of larvae with keratrof. Studia Universitatis Babes-Bolyai Biologia 32:56–59Google Scholar
  16. Douglas AE (2000) Reproductive diapause and the bacterial symbiosis in the sycamore aphid Drepanosiphum platanoides. Ecol Entomol 25:256–261CrossRefGoogle Scholar
  17. Dunn WB, Ellis DI (2005) Metabolomics: current analytical platforms and methodologies. Trends Analyt Chem 24:285–294CrossRefGoogle Scholar
  18. Flannagan RD, Tammariello SP, Joplin KH, Cikra-Ireland RA, Yocum GD, Denlinger DL (1998) Diapause-specific gene expression in pupae of the flesh fly Sarcophaga crassipalpis. Proc Natl Acad Sci USA 95:5616–5620PubMedCrossRefGoogle Scholar
  19. Fields PG, Fleurat-Lessard F, Lavenseau L, Febvay G, Peypelut L, Bonnot G (1998) The effect of cold acclimation and deacclimation on cold tolerance, trehalose and free amino acid levels in Sitophilus granarius and Cryptolestes ferrugineus (Coleoptera). J Insect Physiol 44:955–965PubMedCrossRefGoogle Scholar
  20. Fuchs BC, Bode BP (2006) Stressing out over survival: glutamine as an apoptotic modulator. J Surg Res 131:26–40PubMedCrossRefGoogle Scholar
  21. Goto M, Fujii M, Suzuki K, Sakai M (1998) Factors affecting carbohydrate and free amino acid content in overwintering larvae of Enosima leucotaeniella. J Insect Physiol 44:87–94CrossRefGoogle Scholar
  22. Goto M, Li Y-P, Kayaba S, Outani S, Suzuki K (2001) Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation. J Insect Physiol 47:709–714PubMedCrossRefGoogle Scholar
  23. Hayward SAL, Pavlides SC, Tammariello SP, Rinehart JP, Denlinger DL (2005) Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence. J Insect Physiol 51:631–640PubMedCrossRefGoogle Scholar
  24. Hensgens HESJ, Meijer AJ (1980) Inhibition of urea cycle activity by high concentrations of alanine. Biochem J 186:1–4PubMedGoogle Scholar
  25. Horie Y, Kanda T, Mochida Y (2000) Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori. J Insect Physiol 46:1009–1016PubMedCrossRefGoogle Scholar
  26. Joanisse DR, Storey KB (1995) Temperature acclimation and seasonal responses by enzymes in cold-hardy gall insects. Arch Insect Biochem Physiol 28:339–349CrossRefGoogle Scholar
  27. Kageyama T, Ohnishi E (1971) Carbohydrate metabolism in the eggs of the silkworm Bombyx mori II: anaerobiosis and polyol formation. Dev Growth Differ 15:47–55CrossRefGoogle Scholar
  28. Kaplan F, Kopka J, Haskell DW, Zhao W, Schiller KC, Gatzke N, Sung DY, Guy CL (2004) Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiol 136:4159–4168PubMedCrossRefGoogle Scholar
  29. Kopka J (2006) Current challenges and developments in GC–MS based metabolite profiling technology. J Biotechnol 124:312–322PubMedCrossRefGoogle Scholar
  30. Kruuv J, Glofcheski DJ, Lepock JR (1988) Protective effect of glutamine against freeze-thaw damage in mammalian cells. Cryobiology 25:121–130PubMedCrossRefGoogle Scholar
  31. Kukal O, Denlinger DL, Lee RE Jr (1991) Developmental and metabolic changes induced by anoxia in diapausing and non-diapausing flesh fly pupae. J Comp Physiol [B] 160:683–689Google Scholar
  32. Lee RE (1991) Principles of insect low temperature tolerance. In: Lee RE, Denlinger DL (eds) Insects at low temperatures. Chapman & Hall, New York, pp 17–46Google Scholar
  33. Lee RE, Denlinger DL (1985) Cold tolerance in diapausing and non-diapausing stages of the flesh fly, Sarcophaga crassipalpis. Physiol Entomol 10:309–315Google Scholar
  34. Lee RE, Chen CP, Denlinger DL (1987a) A rapid cold-hardening process in insects. Science 238:1415–1417PubMedCrossRefGoogle Scholar
  35. Lee RE, Chen CP, Meacham MH, Denlinger DL (1987b) Ontogenic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis. J Insect Physiol 33:587–592CrossRefGoogle Scholar
  36. Lee RE, Damodaran K, Yi S-X, Lorigan GA (2006) Rapid cold hardening increases membrane fluidity and cold tolerance of insect cells. Cryobiology 52:459–463PubMedCrossRefGoogle Scholar
  37. Lefevere KS, Koopmanschap AB, De Kort CAD (1989) Changes in the concentrations of metabolites in hemolymph during and after diapause in female Colorado potato beetle Leptinotarsa decemlineata. J Insect Physiol 35:121–128CrossRefGoogle Scholar
  38. Li Y-P, Goto M, Ito S, Sato Y, Sasaki K, Goto N (2001) Physiology of diapause and cold hardiness in the overwintering pupae of the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae) in Japan. J Insect Physiol 47:1181–1187PubMedCrossRefGoogle Scholar
  39. Malmendal A, Overgaard J, Bundy JG, Sørensen JG, Nielsen NC, Loeschcke V, Holmstrup M (2006) Metabolomic profiling of heat stress: hardening and recovery of homeostasis in Drosophila. Am J Physiol Regul Integr Comp Physiol 291:R205–R212PubMedGoogle Scholar
  40. Michaud MR, Denlinger DL (2006) Oleic acid is elevated in cell membranes during rapid cold-hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis. J Insect Physiol 52:1073–1082PubMedCrossRefGoogle Scholar
  41. Morgan TD, Chippendale GM (1983) Free amino acids of the hemolymph of the southwestern corn borer Diatraea grandiosella and the European corn borer Ostrinia nubilalis in relation to their diapause. J Insect Physiol 29:735–740CrossRefGoogle Scholar
  42. Okasaki T, Yamashita O (1981) Changes in glucose and fructose contents during embryonic development of the silkworm Bombyx mori. J Sericult Sci Jpn 50:190–196Google Scholar
  43. Osanai M, Yonezawa Y (1986) Changes in amino-acid pools in the silkworm Bombyx mori during embryonic life: alanine accumulation and its conversion to proline during diapause. Insect Biochem 16:373–380CrossRefGoogle Scholar
  44. Overgaard J, Sorensen JG, Petersen SO, Loeschcke V, Holmstrup M (2005) Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster. J Insect Physiol 51:1173–1182PubMedCrossRefGoogle Scholar
  45. Phanvijhitsiri K, Musch MW, Ropeleski MJ, Chang EB (2005) Molecular mechanisms of L-glutamine modulation of heat stimulated Hsp25 production. FASEB J 19:A1496–A1497Google Scholar
  46. Pullin AS, Wolda H (1993) Glycerol and glucose accumulation during diapause in a tropical beetle. Physiol Entomol 18:75–78Google Scholar
  47. Rinehart JP, Yocum GD, Denlinger DL (2000) Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Sarcophaga crassipalpis. Insect Biochem Mol Biol 30:515–521PubMedCrossRefGoogle Scholar
  48. Rivers DB, Lee RE Jr, Denlinger DL (2000) Cold hardiness of the fly pupal parasitoid Nasonia vitripennis is enhanced by its host, Sarcophaga crassipalpis. J Insect Physiol 46:99–106PubMedCrossRefGoogle Scholar
  49. Salvucci ME (2000) Sorbitol accumulation in whiteflies: evidence for a role in protecting proteins during heat stress. J Therm Biol 25:353–361PubMedCrossRefGoogle Scholar
  50. Schauer N, Steinhauser D, Strelkov S, Schomburg D, Allison G, Moritz T, Lundgrean K, Roessner-Tunali U, Forbes M, Willmitzer L, Fernie AR, Kopka J (2005) GC–MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett 579:1332–1337PubMedCrossRefGoogle Scholar
  51. Slama K, Denlinger DL (1992) Infradian cycles of oxygen consumption in diapausing pupae of the flesh fly, Sarcophaga crassipalpis, monitored by a scanning microrespirographic method. Arch Insect Biochem Physiol 20:135–143PubMedCrossRefGoogle Scholar
  52. So P-W, Fuller BJ (2003) Enhanced energy metabolism during cold hypoxic organ preservation: studies on rat liver after pyruvate supplementation. Cryobiology 46:295–300PubMedCrossRefGoogle Scholar
  53. Stanic B, Jovanovic-Galovic A, Blagojevic DP, Grubor-Lajsic G, Worland R, Spasic MB (2004) Cold hardiness in Ostrinia nubilalis (Lepidoptera: Pyralidae): glycerol content, hexose monophosphate shunt activity, and antioxidative defense system. Eur J Entomol 101:459–466Google Scholar
  54. Storey JM, Storey KB (1983) Regulation of cryoprotectant metabolism in the overwintering gall fly larva Eurosta solidaginis: temperature control of glycerol and sorbitol levels. J Comp Physiol [B] 149:495–502Google Scholar
  55. Storey KB, Storey JM (1986) Freeze tolerant frogs cryoprotectants and tissue metabolism during freeze-thaw cycles. Can J Zool 64:49–56CrossRefGoogle Scholar
  56. Tang X, Pikal MJ (2005) The effects of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze-drying. Pharm Res 22:1167–1175PubMedCrossRefGoogle Scholar
  57. Tomeba H, Oshikiri K, Suzuki K (1988) Changes in the free amino acid pool in the eggs of the emma field cricket Teleogryllus emma (Orthoptera: Gryllidae). Appl Entomol Zool 23:228–233Google Scholar
  58. Touchette BW, Burkholder JM (2000) Overview of the physiological ecology of carbon metabolism in seagrasses. J Exp Mar Biol Ecol 250:169–205PubMedCrossRefGoogle Scholar
  59. Tsumuki H, Kanehisa K (1980) Changes in enzyme activities related to glycerol synthesis in hibernating larvae of the rice stem borer Chilo suppressalis. Appl Entomol Zool 15:285–292Google Scholar
  60. Tsvetkova NM, Quinn PJ (1994) Compatible solutes modulate membrane lipid phase behaviour. In: Cossins AR (ed) Temperature adaptation of biological membranes. Portland Press, London, pp 49–62Google Scholar
  61. Wang H-S, Kang L (2005) Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs. Cryobiology 51:220–229PubMedCrossRefGoogle Scholar
  62. Wang G, Jiang X, Wu L, Li S (2005) Differences in the density, sinking rate and biochemical composition of Centropages tenuiramis (Copepoda: Calanoida) subitaneous and diapause eggs. Mar Ecol Prog Ser 288:165–171CrossRefGoogle Scholar
  63. Weckworth W, Morganthal K (2005) Metabolomics: from pattern recognition to biological interpretation. Drug Discov Today 10:1551–1558CrossRefGoogle Scholar
  64. Yi S-X, Adams TS (2000) Effect of pyriproxyfen and photoperiod on free amino acid concentrations and proteins in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata (Say). J Insect Physiol 46:1341–1353PubMedCrossRefGoogle Scholar
  65. Yi S-X, Bai C (1991) A study in chill-induced glycerol production by Ostrinia furnacalis larvae. Acta Entomol Sin 34:129–134Google Scholar
  66. Zytkovicz TH, Fitzgerald EF, Marsden D, Larson CA, Shih VE, Johnson DM, Strauss AW, Comeau AM, Eaton RB, Grady GF (2001) Tandem mass spectrometric analysis for amino, organic, and fatty acid disorders in newborn dried blood spots: a two-year summary from the New England Newborn Screening Program. Clin Chem 47:1937–1938Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Department of EntomologyOhio State UniversityColumbusUSA

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