Functional analysis of cryoprotectant system in Chymomyza costata

Robert GRGAC

Doctoral thesis

Abstract:

In this study, employing an integrated approach that involves transcriptomics, metabolomics, and other techniques, we aimed to understand the mechanistic roles of the cryoprotective system underlying extreme freeze tolerance in the larvae of the drosophilid fly, Chymomyza costata. Metabolomics unveiled the complex composition of the seasonally accumulated larval innate mixture of putative cryoprotectants. This mixture was predominantly composed of proline and trehalose, supplemented by minor components (glutamine, asparagine, betaine, sarcosine, glycerophospho-choline, and ethanolamine). We identified food ingestion as a significant source for the direct assimilation of amino compounds, while glycogen and phospholipids served as the primary internal sources for the biosynthesis of other cryoprotectants. The utilization of MALDI-MSI to observe cryoprotectants revealed distinct behaviors of proline and trehalose during ecologically relevant, gradual inoculative extracellular freezing of larvae. Trehalose exhibited accumulation in partially dehydrated hemolymph, inducing a transition to the amorphous glass phase. On the other hand, proline migrated to the boundary between extracellular ice and dehydrated hemolymph and tissues, forming a layer of dense viscoelastic liquid. Finally, through a combination of in vivo and in vitro assays, we found that cell membranes are likely targets of freezing injury, while their integrity is sustained by accumulated small cryoprotective molecules and proteins in cold-acclimated C. costata larvae. Contrary to our expectations, our assays did not provide support for the hypothesis that proteins (soluble enzymes) require in vivo stabilization through the accumulation of cryoprotectants.

Abstract:

In this study, employing an integrated approach that involves transcriptomics, metabolomics, and other techniques, we aimed to understand the mechanistic roles of the cryoprotective system underlying extreme freeze tolerance in the larvae of the drosophilid fly, Chymomyza costata. Metabolomics unveiled the complex composition of the seasonally accumulated larval innate mixture of putative cryoprotectants. This mixture was predominantly composed of proline and trehalose, supplemented by minor components (glutamine, asparagine, betaine, sarcosine, glycerophospho-choline, and ethanolamine). We identified food ingestion as a significant source for the direct assimilation of amino compounds, while glycogen and phospholipids served as the primary internal sources for the biosynthesis of other cryoprotectants. The utilization of MALDI-MSI to observe cryoprotectants revealed distinct behaviors of proline and trehalose during ecologically relevant, gradual inoculative extracellular freezing of larvae. Trehalose exhibited accumulation in partially dehydrated hemolymph, inducing a transition to the amorphous glass phase. On the other hand, proline migrated to the boundary between extracellular ice and dehydrated hemolymph and tissues, forming a layer of dense viscoelastic liquid. Finally, through a combination of in vivo and in vitro assays, we found that cell membranes are likely targets of freezing injury, while their integrity is sustained by accumulated small cryoprotective molecules and proteins in cold-acclimated C. costata larvae. Contrary to our expectations, our assays did not provide support for the hypothesis that proteins (soluble enzymes) require in vivo stabilization through the accumulation of cryoprotectants.

Language used: English

Date on which the thesis was submitted / produced: 15. 12. 2023

Thesis defence

• Supervisor: prof. Ing. Vladimír Košťál, CSc.

UNIVERSITY OF SOUTH BOHEMIA IN ČESKÉ BUDĚJOVICE

Faculty of Science

Doctoral programme / field:
Physiology and Immunology / Physiology and Developmental Biology