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The bloodstream formTrypanosoma bruceimaintains essential mitochondrial membrane potential (ΔΨm) through the reverse activity of FoF1-ATP synthase. The ATP that drives this activity is thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). We have shown that this carrier is the only carrier that can import ATP into the mitochondrial matrix to power the FoF1-ATPase. Contrary to expectations, its deletion has no effect on parasite growth, virulence and levels of ΔΨm, suggesting that ATP is produced intramitochondrially by substrate phosphorylation pathways. Therefore, we knocked out the succinyl-CoA synthetase (SCoAS) gene, a key enzyme that produces ATP through substrate phosphorylation. Its absence resulted in changes in the metabolic landscape of the parasite, lower virulence, and reduced mitochondrial ATP content. This minimal mitochondrial ATP pool was maintained by AAC activity as evidenced by the 25- fold increase in sensitivity of the mutant parasites to AAC inhibitor carboxyatractyloside. Under nutrient-limited conditions, suppression of SCoAS expression by RNA interference negatively affected cell growth and levels of ΔΨm. We concluded that the bloodstream mitochondrion is capable of generating ATP via substrate phosphorylation pathways, the importance of which depends on environmental conditions.

Trypanosoma bruceiis the causative agent of the Human and Animal African Trypanosomiases. Mammalian stage parasites infect the bloodstream, central nervous system, but also skin, adipose tissue or lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose which is readily available in mammalian tissues. In addition to glucose, glycerol can be utilised as a carbon and ATP source and also as a substrate for gluconeogenesis. The physiological relevance of glycerol-fed gluconeogenesis for mammalian-infective trypanosome life-cycle stages, however, remins elusive. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase, transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data renders unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and precludes the participation of a novel biochemical pathway involving transaldolase in the process. Sustained metabolic flux in gluconeogenesis in our mutants, including in a triple-null strain, provides new insights into gluconeogenesis and the pentose phosphate pathway, and improves the current understanding of carbon metabolism of the mammalian-infective stages ofT. brucei.

The accumulation of trehalose has been suggested as a mechanism underlying insect cross-tolerance to cold/freezing and drought. Here we show that exposing diapausing larvae of the drosophilid fly, Chymomyza costata to dry conditions significantly stimulates their freeze tolerance. It does not, however, improve their tolerance to desiccation, nor does it significantly affect trehalose concentrations. Next, we use metabolomics to compare the complex alterations to intermediary metabolism pathways in response to three environmental factors with different ecological meanings: environmental drought (an environmental stressor causing mortality), decreasing ambient temperatures (an acclimation stimulus for improvement of cold hardiness), and short days (an environmental signal inducing diapause). We show that all three factors trigger qualitatively similar metabolic rearrangement and a similar phenotypic outcome—improved larval freeze tolerance. The similarities in metabolic response include (but are not restricted to) the accumulation of typical compatible solutes and the accumulation of energy-rich molecules (phosphagens). Based on these results, we suggest that transition to metabolic suppression (a state in which chemical energy demand is relatively low but need for stabilization of macromolecules is high) represents a common axis of metabolic pathway reorganization towards accumulation of non-toxic cytoprotective compounds, which in turn stimulates larval freeze tolerance.

L-amino acids (L-AAs) play different important roles in the physiology of all living organisms. Their chiral counterparts, D-amino acids (D-AAs) are increasingly being recognized as essential molecules in many biological systems. Secondary amino acids with cyclic structures, such as prolines, exhibit conformational rigidity and thus unique properties in the structural and protein folding. Despite their widespread occurrence, much less attention was paid to their chiral analysis, particularly when the minor, typically D-enantiomer, is present in low amounts in a complex biological matrix. In this paper, a cost-effective, chiral GC-MS method is described for capillary Chirasil-L-Val separation of nine cyclic secondary amino acid enantiomers with four-, five-, and six-membered rings, involving azetidine-2-carboxylic acid, pipecolic acid, nipecotic acid, proline, isomeric cis/trans 3-hydroxy, 4-hydroxyproline, and cis/trans-5-hydroxy-L-pipecolic acid in the excess of its enantiomeric antipode. The sample preparation involves in-situ derivatization with heptafluorobutyl chloroformate, simultaneous liquid-liquid micro-extraction into isooctane followed by amidation of the arising low-polar derivatives with methylamine, an evaporation step, re-dissolution, and final GC-MS analysis. The developed method was used for analyses of human biofluids, biologically active peptides containing chiral proline constituents, and collagen.

Dermanyssus gallinae is a blood-feeding mite that parasitises on wild birds and farmed poultry. The D. gallinae mite has a short life cycle of fewer than two weeks from the egg to an egg-laying female. The remarkably swift processing of blood, together with the capacity to blood-feed in most developmental stages, makes this mite a highly debilitating pest. We have constructed developmental stage-specific transcriptomes, through Illumina RNA-seq, to mine the repertoire of protein-encoding mRNA transcripts, products of which participate in key processes that ensure the success of blood digestion, rapid ontogeny, and immunity. As a result of high reproductive capacity, the prevalence of D. gallinae in egg-producing poultry farms globally causes significant economic losses. Acaricides that are used to limit the reproduction of D. gallinae mites target cys-loop ion channels, which are widely shared across the phylogeny of invertebrates. To catalogue a comprehensive list of potential invertebrate-specific ion channels, we have constructed and analysed an additional RNA-seq library of D. gallinae micro-dissected midguts, a tissue with direct exposure to host blood and potential anti-parasitics. We phylogenetically defined groups of cys-loop proteins and probed their sensitivity to selected acaricides. Ultimately, we have catalogued all assembled transcripts and their expression values in a hyper-linked excel sheet with available sequences of individual contigs. The transcriptomic data were complemented by mass-spectrometry (MS)-based metabolite identification and by viability assays using selected inhibitors applied either by microinjection or through artificial membrane feeding. Additionally, we have described the RNA-virome of D. gallinae and identified a novel virus dubbed Red Mite Quaranjavirus 1.

Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on mechanisms of how the individual CPs interact, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata that survives long-term cryopreservation in liquid nitrogen. The CPs (proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine, and sarcosine) accumulate in hemolymph in a ratio of 313:108:55:26:6:4:3:0.5 mmol.L-1. Using calorimetry, we show that the artificial mixtures, mimicking the concentrations of major CPs’ in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using MALDI-MSI, we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it likely forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.Summary statementThe composition of natural cryoprotectant mixture of the extremely freeze-tolerant insect is revealed. Components of the mixture work in synergy and behave differently during organismal freezing and cryopreservation.

Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular K+concentration ([K+]). Cold-induced depolarization is linked to cold injury in chill-susceptible insects, and the locust, Locusta migratoria, has been shown to improve cold tolerance following cold acclimation through depolarization resistance. Here we investigate how cold acclimation influences depolarization resistance and how this resistance relates to improved cold tolerance. To address this question, we investigated if cold acclimation affects the electrogenic transport capacity and/or the relative K+permeability during cold exposure by measuring membrane potentials of warm- and cold-acclimated locusts in the presence and absence of ouabain (Na+-K+pump blocker) or 4-aminopyridine (4-AP; voltage-gated K+channel blocker). In addition, we compared the membrane lipid composition of muscle tissue from warm- and cold-acclimated locust and the abundance of a range transcripts related to ion transport and cell injury accumulation. We found that cold-acclimated locusts are depolarization resistant due to an elevated K+permeability, facilitated by opening of 4-AP-sensitive K+channels. In accordance, cold acclimation was associated with an increased abundance of Shaker transcripts (gene encoding 4-AP-sensitive voltage-gated K+channels). Furthermore, we found that cold acclimation improved muscle cell viability following exposure to cold and hyperkalemia even when muscles were depolarized substantially. Thus cold acclimation confers resistance to depolarization by altering the relative ion permeability, but cold-acclimated locusts are also more tolerant to depolarization.

The protection of carboxyl groups by esterification has been the most common method in macroscale and microscale chemistries. The esterification is usually conducted under anhydrous conditions; however, in biological chemistry and related fields, the reaction is of major concern in aqueous environments. Immediate esterification of the carboxyl in aqueous alcoholic media driven by an alkyl chloroformate and pyridine has been such a method which has found widespread use in many research and industrial laboratories. Nevertheless, the reaction mechanism has not yet been investigated, to our knowledge, and is not well understood. Herein, we describe the reaction intermediates and demonstrate that the reaction proceeds via a continual formation of the N-acylpyridinium intermediate decomposed by several reaction channels to the final ester. The understanding of the mechanism could encourage novel laboratory applications of this important esterification method.

Gemmatimonas phototrophica AP64 is the first phototrophic representative of the bacterial phylum Gemmatimonadetes. The cells contain photosynthetic complexes with bacteriochlorophyll a as the main light-harvesting pigment and an unknown carotenoid with a single broad absorption band at 490 nm in methanol. The carotenoid was extracted from isolated photosynthetic complexes, and purified by liquid chromatography. A combination of nuclear magnetic resonance (1H NMR, COSY, 1H-13C HSQC, 1H-13C HMBC, J-resolved, and ROESY), high-resolution mass spectroscopy, Fourier-transformed infra-red, and Raman spectroscopy was used to determine its chemical structure. The novel linear carotenoid, that we have named gemmatoxanthin, contains 11 conjugated double bonds and is further substituted by methoxy, carboxyl and aldehyde groups. Its IUPAC-IUBMB semi-systematic name is 1′-Methoxy-19′-oxo-3′,4′-didehydro-7,8,1′,2′-tetrahydro- Ψ, Ψ carotene-16-oic acid. To our best knowledge, the presence of the carboxyl, methoxy and aldehyde groups on a linear C40 carotenoid backbone is reported here for the first time.

Background The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. Results We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. Conclusions The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

Several families of 3,4-dimethylthiolane-based compounds spontaneously formed upon cutting of onion (Allium cepa) were studied. We report the isolation of the first known example of a naturally occ...

Extracellular freezing of insect body water may cause lethal injury either by direct mechanical stress exerted by growing ice crystals on cells and tissues or, indirectly, by deleterious physico-chemical effects linked to freeze-induced cell dehydration. Here we present results showing that the macroscopic damage (cell ruptures, tissue disintegration) to fat body of Drosophila melanogaster is not directly caused by mechanical forces linked to growth of ice crystals but rather represents a secondary consequence of other primary freeze injuries occurring at subcellular or microscopic levels. Larvae of D. melanogaster were acclimated to produce variants ranging from freeze susceptible to freeze tolerant. Then, larvae were exposed to supercooling and freezing stresses at different subzero temperatures. The larval survival and macroscopic damage to fat body tissue was scored in 1632 larvae exposed to cold stress. In most cases, fat body damage was not evident immediately following cold stress but developed later. This suggests that the fat body disintegration is a consequence rather than a cause of cold injury. Analysis of fat body membrane phospholipids revealed that increased freeze tolerance was associated with increased relative proportion of phosphatidylethanolamines (PEs) at the expense of phosphatidylcholines (PCs). The PE/PC ratio increased from 1.08 in freeze-susceptible larvae to 2.10 in freeze-tolerant larvae. The potential effects of changing PE/PC ratio on phospholipid bilayer stability upon supercooling and freezing stress are discussed.

L-amino acids (L-AAs) play different important roles in the physiology of all living organisms. Their chiral counterparts, D-amino acids (D-AAs) are increasingly being recognized as essential molecules in many biological systems. Secondary amino acids with cyclic structures, such as prolines, exhibit conformational rigidity and thus unique properties in the structural and protein folding. Despite their widespread occurrence, much less attention was paid to their chiral analysis, particularly when the minor, typically D-enantiomer, is present in low amounts in a complex biological matrix. In this paper, a cost-effective, chiral GC-MS method is described for capillary Chirasil-L-Val separation of nine cyclic secondary amino acid enantiomers with four-, five-, and six-membered rings, involving azetidine-2-carboxylic acid, pipecolic acid, nipecotic acid, proline, isomeric cis/trans 3-hydroxy, 4-hydroxyproline, and cis/trans-5-hydroxy-L-pipecolic acid in the excess of its enantiomeric antipode. The sample preparation involves in-situ derivatization with heptafluorobutyl chloroformate, simultaneous liquid-liquid micro-extraction into isooctane followed by amidation of the arising low-polar derivatives with methylamine, an evaporation step, re-dissolution, and final GC-MS analysis. The developed method was used for analyses of human biofluids, biologically active peptides containing chiral proline constituents, and collagen.

Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular K

Diaspores of myrmecochorous plants consist of a seed (or fruit) and an attached appendage (elaiosome) which attracts ants. The elaiosome is a food resource for ants, whereas the seed is an energy source for subsequent germination and plant establishment. Although myrmecochory occurs in many phylogenetically unrelated lineages, multiple phylogenetic lineages display similar variation in elaiosome and seed metabolite composition due to convergent evolution. We focused on four families (Amaryllidaceae, Boraginaceae, Papaveraceae and Poaceae) each represented by two species from different genera. Diaspores of three populations per species were sampled and concentrations of 60 metabolites from five groups (amino acids, fatty acids, organic acids, polyols and sugars) were determined for both elaiosomes and seeds. Variability in metabolite composition was decomposed by hierarchical ANOVA and variation partitioning using redundancy analysis (reflecting both species nested within families, crossed with seed vs. elaiosome). Differences in the metabolite composition of elaiosomes and seeds were consistent across multiple phylogenetic origins (with more pronounced differences at the level of individual metabolites than at the level of metabolite groups) and supported the idea of convergent evolution under strong selection pressure. Elaiosomes contained higher amounts of easily digestible metabolites (especially amino acids) than seeds. Fatty acids were not more concentrated in elaiosomes, which contradicts the literal translation of "elaiosome" (= oil body). The differentiation of metabolite composition closely reflected taxonomic relatedness, particularly at the family level. Differences among populations within species were small, so the metabolite composition can thus be considered as a trait with relatively low intraspecific variability.

The compounds responsible for the bitter off-taste of processed onion (Allium cepa) were studied. Using a series of sensory-guided HPLC fractionations, the existence of nine groups of hitherto unknown sulfur compounds has been revealed. On the basis of spectroscopic data (MS, NMR, and IR), it was found that these compounds, trivially named allithiolanes A–I, are members of a large family of structurally closely related derivatives of 3,4-dimethylthiolane S-oxide, with the general formulas of CxHyO2S4, CxHyO3S5, and CxHyO4S6 (x = 10–18, y = 18–30). The presence of multiple stereoisomers was observed for each group of allithiolanes. Allithiolanes possess an unpleasantly bitter taste with detection thresholds in the range of 15–30 ppm. Formation pathways of these newly discovered sulfur compounds were proposed.

Physiological adjustments accompanying insect cold acclimation prior to cold stress have been relatively well explored. In contrast, recovery from cold stress received much less attention. Here we report on recovery of drosophilid fly larvae (Chymomyza costata) from three different levels of cold stress: supercooling to −10 °C, freezing at −30 °C, and cryopreservation at −196 °C. Analysis of larval CO2 production suggested that recovery from all three cold stresses requires access to additional energy reserves to support cold-injury repair processes. Metabolomic profiling (targeting 41 metabolites using mass spectrometry) and custom microarray analysis (targeting 1,124 candidate mRNA sequences) indicated that additional energy was needed to: clear by-products of anaerobic metabolism, deal with oxidative stress, re-fold partially denatured proteins, and remove damaged proteins, complexes and/or organelles. Metabolomic and transcriptomic recovery profiles were closely similar in supercooled and frozen larvae, most of which successfully repaired the cold injury and metamorphosed into adults. In contrast, the majority of cryopreseved larvae failed to proceed in ontogenesis, showed specific metabolic perturbations suggesting impaired mitochondrial function, and failed to up-regulate a set of 116 specific genes potentially linked to repair of cold injury.

Escalation (macroevolutionary increase) or divergence (disparity between relatives) in trait values are two frequent outcomes of the plant-herbivore arms race. We studied the defences and caterpillars associated with 21 sympatric New Guinean figs. Herbivore generalists were concentrated on hosts with low protease and oxidative activity. The distribution of specialists correlated with phylogeny, protease and trichomes. Additionally, highly specialised Asota moths used alkaloid rich plants. The evolution of proteases was conserved, alkaloid diversity has escalated across the studied species, oxidative activity has escalated within one clade, and trichomes have diverged across the phylogeny. Herbivore specificity correlated with their response to host defences: escalating traits largely affected generalists and divergent traits specialists; but the effect of escalating traits on extreme specialists was positive. In turn, the evolution of defences in Ficus can be driven towards both escalation and divergence in individual traits, in combination providing protection against a broad spectrum of herbivores.

The use of porous polymer materials for vascular prostheses demonstrates promising results. Fleece-like non-woven structures can be generated by atomization of dissolved polycarbonate urethanes. This article focuses on the manufacturing process for the fleece structures. The solution is atomized through a high-volume low-pressure nozzle. The solvent evaporates during time of flight to target, so that small fibres are formed from each drop of solution. By using different rotating molds and positioning systems, tubular shapes or open surfaces can be generated. The manufacturing process is described in detail. The usability of different grades of polycarbonate urethane is tested and the time necessary for evaporation of the solvent from the mesh is assessed. The influence of several basic parameters on the mechanical properties of the generated non-woven material is analysed and discussed.

Numerous industrial organic pollutants such as aromates, alkoxyalcohols, other organic solvents and monomers are absorbed, metabolized, and finally excreted in urine mostly as carboxylic acids that are determined as biomarkers of exposure. For a number of these xenometabolites, biological limits (levels of biomarkers in biological material) have been established to prevent damage to human health. Till now, most of the analytical procedures used have been optimized for one or a few analytes. Here, we report a more comprehensive approach enabling rapid GC-MS screening of sixteen acidic biomarkers in urine that are metabolized in the human body from several important industrial chemicals; benzene, toluene, styrene, xylenes, alkoxyalcohols, carbon disulfide, furfural and N,N-dimethylformamide. The new method involves immediate in situ derivatization - liquid liquid microextraction of urine by an ethyl chloroformate-ethanol-chloroform-pyridine medium and GC-MS analysis of the derivatized analytes in the lower organic phase. The xenometabolite set represents diverse chemical structures and some of hippuric and mercapturic acids also provided unusual derivatives that were unambiguously elucidated by means of new ethyl chloroformates labeled with stable isotopes and by synthesis of the missing reference standards. In the next step, an automated routine was developed for GC-MS/MS analysis using a MetaboAuto® sample preparation workstation and the new method was validated for fourteen metabolites over the relevant concentration range of each analyte in the spiked pooled human urine. It shows good linearity (R2 ≥ 0.982), accuracy (from 85% to 120%), precision (from 0.7% to 20%) and recovery (from 89% to 120%). The method performance was further successfully proved by GC-MS/MS analysis of the certified IP45 and RM6009 reference urines. Moreover, we show that the new method opens up the possibility for biomonitoring of combined and cumulative occupational exposures as well as for urinary metabolite profiling of persons exposed to harmful industrial chemicals.

Diplonemids belong to the most diverse and abundant marine protists, which places them among the key players of the oceanic ecosystem. Under in vitro conditions, their best-known representative Diplonema papillatum accumulates in its cytoplasm a crystalline polymer. When grown under the nutrient-poor conditions, but not nutrient-rich conditions, D. papillatum synthesizes a β-1,3-glucan polymer, also known as paramylon. This phenomenon is unexpected, as it is in striking contrast to the accumulation of paramylon in euglenids, since these related flagellates synthesize this polymer solely under nutrient-rich conditions. The capacity of D. papillatum to store an energy source in the form of polysaccharides when the environment is poor in nutrients is unexpected and may contribute to the wide distribution of these protists in the ocean.

The risks of depletion of energy reserves and encountering lethally low temperatures are considered as two important mortality factors that may limit winter survival of mosquito, Culex pipiens f. pipiens populations. Here we show that the autumn females carry lipid reserves, which are safely sufficient for at least two overwintering periods, provided the females diapausing at temperatures typical for underground spaces (0 °C - 8 °C) would continuously rest at a standard metabolic rate (SMR). The overwintering females, however, switch from SMR to much higher metabolic rate during flight, either seeking for optimal microhabitat within the shelter or in response to disturbances by air current or predator attack. These behaviors result in fast oxidation of lipid reserves and, therefore, the autumn load of energy reserves may actually limit winter survival under specific circumstances. Next, we show that the level of females' cold hardiness is physiologically set relatively weak for overwintering in open field, above-ground habitats, but is ecologically entirely sufficient for overwintering in most underground spaces. The characteristics of suitable overwintering shelters are: no or limited risk of contact with ice crystals, no or limited air movements, winter temperatures relatively stable between +2 and + 6 °C, winter minimum does not drop below −4 °C for longer than one week, or below −8 °C for longer than 1 day.

Low temperatures limit the distribution and abundance of ectotherms. However, many insects can survive low temperatures by employing one of two cold tolerance strategies: freeze avoidance or freeze tolerance. Very few species can employ both strategies, but those that do provide a rare opportunity to study the mechanisms that differentiate freeze tolerance and freeze avoidance. We showed that overwintering pupae of the cabbage white butterfly Pieris rapae can be freeze tolerant or freeze avoidant. Pupae from a population of P. rapae in northeastern Russia (Yakutsk) froze at c. −9.3 °C and were freeze-tolerant in 2002–2003 when overwintered outside. However, P. rapae from both Yakutsk and southern Canada (London) acclimated to milder laboratory conditions in 2014 and 2017 froze at lower temperatures (< −20 °C) and were freeze-avoidant. Summer-collected P. rapae larvae (collected in Yakutsk in 2016) were partially freeze-tolerant, and decreased the temperature at which they froze in response to starvation at mild low temperatures (4 °C) and repeated partial freezing events. By comparing similarly-acclimated P. rapae pupae from both populations, we identified molecules that may facilitate low temperature tolerance, including the hemolymph ice-binding molecules and several potential low molecular weight cryoprotectants. Pieris rapae from Yakutsk exhibited high physiological plasticity, accumulating cryoprotectants and almost doubling their hemolymph osmolality when supercooled to −15 °C for two weeks, while the London P. rapae population exhibited minimal plasticity. We hypothesize that physiological plasticity is an important adaptation to extreme low temperatures (i.e. in Yakutsk) and may facilitate the transition between freeze avoidance and freeze tolerance.

The biochemical and molecular mechanisms underlying insect cold acclimation prior to cold stress are relatively well explored, but the mechanisms linked to recovery and repair after cold stress have received much less attention. Here we focus on recovery from cold stress in the larvae of the vinegar fly (Drosophila melanogaster) that were exposed to two physiologically distinct cold stress situations: supercooling (S, survival > 95%) and freezing (F, survival < 10%), both at -5 °C. We analysed the metabolic and transcriptomic responses to cold stress via GC-MS/LC-MS and whole-genome microarrays, respectively. Both stresses (S and F) caused metabolic perturbations which were transient in supercooled larvae but deeper and irreversible in frozen larvae. Differential gene expression analysis revealed a clear disparity in responses to supercooling and freezing (less than 10% of DE genes overlapped between S and F larvae). Using GO term enrichment analysis and KEGG pathway mapping, we identified the stimulation of immune response pathways as a strong candidate mechanism for coping with supercooling. Supercooling caused complex transcriptional activation of innate immunity potential: from Lysozyme-mediated degradation of bacterial cell walls, recognition of pathogen signals, through phagocytosis and lysosomal degradation, Toll and Imd signaling, to upregulation of genes coding for different antimicrobial peptides. The transcriptomic response to freezing was instead dominated by degradation of macromolecules and death-related processes such as autophagy and apoptosis. Of the 45 upregulated DE genes overlapping in responses to supercooling and freezing, 26 were broadly ascribable to defense and repair functions.

Some insects rely on the strategy of freeze tolerance for winter survival. During freezing, extracellular body water transitions from the liquid to solid phase and cells undergo freeze-induced dehydration. Here we present results of a thermal analysis (from differential scanning calorimetry) of ice fraction dynamics during gradual cooling after inoculative freezing in variously acclimated larvae of two drosophilid flies, Drosophila melanogaster and Chymomyza costata. Although the species and variants ranged broadly between 0 and close to 100% survival of freezing, there were relatively small differences in ice fraction dynamics. For instance, the maximum ice fraction (IFmax) ranged between 67.9 and 77.7% total body water (TBW). The C. costata larvae showed statistically significant phenotypic shifts in parameters of ice fraction dynamics (melting point and IFmax) upon entry into diapause, cold-acclimation, and feeding on a proline-augmented diet. These differences were mostly driven by colligative effects of accumulated proline (ranging between 6 and 487 mmol.kg−1 TBW) and other metabolites. Our data suggest that these colligative effects per se do not represent a sufficient mechanistic explanation for high freeze tolerance observed in diapausing, cold-acclimated C. costata larvae. Instead, we hypothesize that accumulated proline exerts its protective role via a combination of mechanisms. Specifically, we found a tight association between proline-induced stimulation of glass transition in partially-frozen body liquids (vitrification) and survival of cryopreservation in liquid nitrogen.

The ectoparasitoid wasp, Nasonia vitripennis can enhance its cold tolerance by exploiting a maternally-induced larval diapause. A simple manipulation of the fly host diapause status and supplementation of the host diet with proline also dramatically increase cold tolerance in the parasitoid. In this study, we used a metabolomics approach to define alterations in metabolite profiles of N. vitripennis caused by diapause in the parasitoid, diapause of the host, and augmentation of the host's diet with proline. Metabolic profiles of diapausing and nondiapausing parasitoid were significantly differentiated, with pronounced distinctions in levels of multiple cryoprotectants, amino acids, and carbohydrates. The dynamic nature of diapause was underscored by a shift in the wasp's metabolomic profile as the duration of diapause increased, a feature especially evident for increased concentrations of a suite of cryoprotectants. Metabolic pathways involved in amino acid and carbohydrate metabolism were distinctly enriched during diapause in the parasitoid. Host diapause status also elicited a pronounced effect on metabolic signatures of the parasitoid, noted by higher cryoprotectants and elevated compounds derived from glycolysis. Proline supplementation of the host diet did not translate directly into elevated proline in the parasitoid but resulted in an alteration in the abundance of many other metabolites, including elevated concentrations of essential amino acids, and reduction in metabolites linked to energy utilization, lipid and amino acid metabolism. Thus, the enhanced cold tolerance of N. vitripennis associated with proline augmentation of the host diet appears to be an indirect effect caused by the metabolic perturbations associated with diet supplementation.

The bean bug (Riptortus pedestris) is a pest of soybeans and other legumes in Japan and other Asian countries. It enters a facultative adult diapause on exposure to short days. While photoperiodism and diapause are well understood in R. pedestris, knowledge of cold tolerance is very limited, as is information on the effect of diapause on cold tolerance. We examined the effect of photoperiod, cold acclimation, and feeding status on cold tolerance in R. pedestris. We found that cold acclimation significantly increased survival at −10°C in both long- and short-day adult R. pedestris. Since the difference in cold survival between long- and short-day cold-acclimated groups was only marginal, we conclude that entering diapause is not crucial for R. pedestris to successfully pass through cold acclimation and become cold tolerant. We observed similar effects in 5th instar nymphs, with both long- and short-day cold-acclimated groups surviving longer cold exposures compared with non-acclimated groups. Starvation, which was tested only in adult bugs, had only a negligible and negative impact on cold survival. Although cold tolerance significantly increased with cold acclimation in adult bugs, supercooling capacity unexpectedly decreased. Our results suggest that changes in supercooling capacity as well as in water content are unrelated to cold tolerance in R. pedestris. An analysis of metabolites revealed differences between the treatments, and while several metabolites markedly increased with cold acclimation, their concentrations were too low to have a significant effect on cold tolerance.

Diapause is a deep resting stage facilitating temporal avoidance of unfavourable environmental conditions, and is used by many insects to adapt their life cycle to seasonal variation. Although considerable work has been invested in trying to understand each of the major diapause stages (induction, maintenance and termination), we know very little about the transitions between stages, especially diapause termination. Understanding diapause termination is crucial for modelling and predicting spring emergence and winter physiology of insects, including many pest insects. In order to gain these insights, we investigated metabolome dynamics across diapause development in pupae of the butterfly

The alveolate algae Chromera velia and Vitrella brassicaformis (chromerids) are the closest known phototrophic relatives to apicomplexan parasites. Apicomplexans are responsible for fatal diseases of humans and animals and severe economic losses. Availability of the genome sequences of chromerids together with easy and rapid culturing of C. velia makes this alga a suitable model for investigating elementary biochemical principals potentially important for the apicomplexan pathogenicity. Such knowledge allows us to better understand processes during the evolutionary transition from a phototrophy to the parasitism in Apicomplexa. We explored lipidomes of both algae using high-performance liquid chromatography with mass spectrometry or gas chromatography with flame ionization detection. A single high-performance liquid chromatography with mass spectrometry analysis in both ionization modes was sufficient for the separation and semi-quantification of lipids in chromerid algae. We detected more than 250 analytes belonging to five structural lipid classes, two lipid classes of precursors and intermediates, and triacylglycerols as storage lipids. Identification of suggested structures was confirmed by high-resolution mass spectrometry with an Orbitrap mass analyzer. An outstandingly high accumulation of storage triacylglycerols was found in both species. All the investigated aspects make C. velia a prospective organism for further applications in biotechnology. This article is protected by copyright. All rights reserved

Diapause is a deep resting stage facilitating temporal avoidance of unfavourable environmental conditions that is used by many insects to adapt their life cycle to seasonal variation. Although considerable work has been invested in trying to understand each of the major diapause stages (induction, maintenance and termination), we know very little about the transitions between stages, especially diapause termination. Understanding diapause termination is critical for modelling and predicting spring emergence and winter physiology of insects, including many pest insects. In order to gain these insights we investigated metabolome dynamics across diapause development in pupae of the butterfly Pieris napi, which exhibits adaptive latitudinal variation in the length of endogenous diapause that is uniquely well characterized. By employing a time-series experiment we show that the whole-body metabolome is highly dynamic throughout diapause and differs between pupae kept at a diapause-terminating (low), or at a diapause-maintaining (high) temperature. We show major physiological transitions through diapause, separated temperature-dependent from temperature-independent processes and identified significant patterns of metabolite accumulation and degradation. Together the data show that while the general diapause phenotype (suppressed metabolism, increased cold tolerance) is established in a temperature-independent fashion, diapause termination is temperature-dependent and requires a cold signal. This revealed several metabolites that are only accumulated in diapause terminating conditions and degraded in a temperature-unrelated fashion during diapause termination. In conclusion, our findings indicate that some metabolites, in addition to functioning as e.g. cryoprotectants, are candidates for having regulatory roles as metabolic clocks or time-keepers during diapause.

The cryopreservation techniques proposed for embryos of the fruit fly Drosophila melanogaster are not yet ready for practical use. Alternative methods for long-term storage of D. melanogaster strains, although urgently needed, do not exist. Herein, we describe a narrow interval of low temperatures under which the larvae of D. melanogaster can be stored in quiescence for up to two months. The development of larvae was arrested at the pre-wandering stage under fluctuating thermal regime (FTR), which simultaneously resulted in diminishing the accumulation of indirect chill injuries. Our physiological, metabolomic and transcriptomic analyses revealed that compared to larvae stored at constant low temperatures, the larvae stored under FTR conditions were able to decrease the rates of depletion of energy substrates, exploited brief warm episodes of FTR for homeostatic control of metabolite levels and more efficiently exerted protection against oxidative damage.

The fruit fly Drosophila melanogaster is an insect of tropical origin. Its larval stage is evolutionarily adapted for rapid growth and development under warm conditions and shows high sensitivity to cold. In this study, we further developed an optimal acclimation and freezing protocol that significantly improves larval freeze tolerance (an ability to survive at −5°C when most of the freezable fraction of water is converted to ice). Using the optimal protocol, freeze survival to adult stage increased from 0.7% to 12.6% in the larvae fed standard diet (agar, sugar, yeast, cornmeal). Next, we fed the larvae diets augmented with 31 different amino compounds, administered in different concentrations, and observed their effects on larval metabolomic composition, viability, rate of development and freeze tolerance. While some diet additives were toxic, others showed positive effects on freeze tolerance. Statistical correlation revealed tight association between high freeze tolerance and high levels of amino compounds involved in arginine and proline metabolism. Proline- and arginine-augmented diets showed the highest potential, improving freeze survival to 42.1% and 50.6%, respectively. Two plausible mechanisms by which high concentrations of proline and arginine might stimulate high freeze tolerance are discussed: (i) proline, probably in combination with trehalose, could reduce partial unfolding of proteins and prevent membrane fusions in the larvae exposed to thermal stress (prior to freezing) or during freeze dehydration; (ii) both arginine and proline are exceptional among amino compounds in their ability to form supramolecular aggregates which probably bind partially unfolded proteins and inhibit their aggregation under increasing freeze dehydration.