Cryoprotective Metabolites Are Sourced from Both External Diet and Internal Macromolecular Reserves during Metabolic Reprogramming for Freeze Tolerance in Drosophilid Fly, Chymomyza costata.

Martin Moos,Robert Grgac,Stanislav Opekar,Petr Šimek,Vladimír Koštál,Jaroslava Korbelová,Tomáš Štětina

doi:10.3390/metabo12020163

Abstract

Many cold-acclimated insects accumulate high concentrations of low molecular weight cryoprotectants (CPs) in order to tolerate low subzero temperatures or internal freezing. The sources from which carbon skeletons for CP biosynthesis are driven, and the metabolic reprogramming linked to cold acclimation, are not sufficiently understood. Here we aim to resolve the metabolism of putative CPs by mapping relative changes in concentration of 56 metabolites and expression of 95 relevant genes as larvae of the drosophilid fly, Chymomyza costata transition from a freeze sensitive to a freeze tolerant phenotype during gradual cold acclimation. We found that C. costata larvae may directly assimilate amino acids proline and glutamate from diet to acquire at least half of their large proline stocks (up to 55 µg per average 2 mg larva). Metabolic conversion of internal glutamine reserves that build up in early diapause may explain the second half of proline accumulation, while the metabolic conversion of ornithine and the degradation of larval collagens and other proteins might be two additional minor sources. Next, we confirm that glycogen reserves represent the major source of glucose units for trehalose synthesis and accumulation (up to 27 µg per larva), while the diet may serve as an additional source. Finally, we suggest that interconversions of phospholipids may release accumulated glycero-phosphocholine (GPC) and -ethanolamine (GPE). Choline is a source of accumulated methylamines: glycine-betaine and sarcosine. The sum of methylamines together with GPE and GPC represents approximately 2 µg per larva. In conclusion, we found that food ingestion may be an important source of carbon skeletons for direct assimilation of, and/or metabolic conversions to, CPs in a diapausing and cold-acclimated insect. So far, the cold-acclimation- linked accumulation of CPs in insects was considered to be sourced mainly from internal macromolecular reserves.

Highlights

Convergent evolution repeatedly ‘discovered’ benefits of accumulation of a stereotypic family of cytoprotectant molecules in various organisms from archaea and bacteria to animals responding to different environmental stressors such as heat, cold, freezing, drought, hypersalinity, or high hydrostatic pressure [1–4]
We focus on distinguishing between alternative sources of carbon skeletons for the CPs’ biosynthesis, as follows. (i) Proline: is it derived from stored macromolecular reserves such as collagens and total proteins or from metabolic precursors such as glutamine, glutamate and ornithine, or rather assimilated from diet as an external source? (ii) Trehalose: derived from glycogen reserves or dietary starch and glucose? (iii) Methylamines: choline coming from interconversions of phospholipids or glycine which can be massively released during the breakdown of collagens?
Our results suggest that high concentrations of proline in SDA larvae of C. costata originate from different sources: (i) direct assimilation of ingested dietary proline, together with metabolic conversion of dietary glutamate, can explain about one half of the proline accumulation; (ii) metabolic conversion of larval glutamine reserves formed in early diapause can explain the second half of proline accumulation; (iii) two other minor sources can be the metabolic conversion of ornithine and the degradation of larval extracellular matrix (ECM) collagens and other proteins

Summary

Introduction

Convergent evolution repeatedly ‘discovered’ benefits of accumulation of a stereotypic family of cytoprotectant molecules (sugars and polyols, amino acids and derivatives, methylamines) in various organisms from archaea and bacteria to animals responding to different environmental stressors such as heat, cold, freezing, drought, hypersalinity, or high hydrostatic pressure [1–4]. Whatever strategy the insect uses, the accumulation of CPs is considered to be one of the major mechanisms that protects insect cells against damage exerted by low temperatures (supercooling), growing ice crystals and freeze-dehydration (freeze tolerance), or evaporative loss of body water (cryoprotective dehydration) [5–8]. We contribute by tracing the sources of carbon skeletons and the pathways for biosynthesis of all three CP classes in the extremely freeze-tolerant drosophilid fly, Chymomyza costata. The larvae of this fly survive winter in cold-temperate and sub-arctic climate zones in a state of deep dormancy (diapause), during which they acquire extremely high levels of freeze tolerance. The diapause cold acclimated larvae can even survive long-term cryopreservation in liquid nitrogen (LN2), which makes them an interesting model for cryobiology [19–22]

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