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

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.

Consuming Royal Jelly Causes Mosquitoes to Shift Into and Out of Their Overwintering Dormancy

“…Additionally, we found choline is upregulated in diapausing mosquitoes. Choline is predicted to function as a cryoprotectant derived from the diet (Moos et al, 2022), which would serve a critical role in withstanding the low temperatures associated with winter.…”

Insect cell plasma membranes do, while soluble enzymes do not, need stabilization by accumulated cryoprotectant molecules during freezing stress

“…100 and 160 mg.mL −1 for freeze-sensitive and -tolerant phenotypes, respectively). Drosophilid larvae accumulate serum proteins late in their ontogeny (prior to metamorphosis) (53) and many insects, including larvae of C. costata , do accumulate even more of them prior to diapause, as a source of amino acids for post-diapause development (54, 55). The BSA and other membrane-non-permeable compounds such as saccharose, Ficoll, PEG, or hydroxyethyl starch are often added to cryopreservation solutions for mammalian sperm (56) where they likely help to maintain membrane integrity indirectly via affecting the thermal transitions in extracellular medium including kinetics of growth and morphology of ice crystals and glass transition temperatures (57, 58), and/or by reducing the membrane lipid peroxidation by reactive oxygen species (59).…”

Insect cross-tolerance to freezing and drought stress: role of metabolic rearrangement

“…The larvae were weighed to obtain fresh mass, plunged into liquid nitrogen, and stored at −80°C until analysis. Details on extraction of metabolites from larval samples and analysis using four different mass spectrometry-based (MS) analytical platforms as described previously [42,48] are shown in the electronic supplementary material.…”