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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.

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.

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 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.

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 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.

Methanolic extract from lyophilized roots of Impatiens glandulifera Royle was analyzed by high performance liquid chromatography using DAD and FLD detection and this revealed one dominant highly fluorescent very unstable substance. The stability of this derivative is strongly dependent on the plant material drying procedure and extraction procedure used. The structure of the substance was established as 1,2,4-trihydroxynaphthalene-1-O-glucoside (THNG) according LC-MS and NMR measurements. When lyophilized plant material was extracted with methanol an almost four times higher amount of THNG was found in the extract, compared to the amount of 2-hydroxy-1,4-naphthoquinone obtained, while in the case of the same lyophilized plant material extracted with water there was no THNG in the extract. The main compounds in this case was 2-hydroxy-1,4-naphthoquinone. In the plant material dried at the laboratory temperature and extracted by methanol there are only traces of THNG.

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.