Transcriptome and Proteome Analyses Reveal Stage-Specific DNA Damage Response in Embryos of Sturgeon (Acipenser ruthenus).

DNA damage during early life stages may have a negative effect on embryo development, inducing mortality and malformations that have long-lasting effects during adult life. Therefore, in the current study, we analyzed the effect of DNA damage induced by genotoxicants (camptothecin (CPT) and olaparib) at different stages of embryo development. The survival, DNA fragmentation, transcriptome, and proteome of the endangered sturgeon Acipenser ruthenus were analyzed. Sturgeons are non-model fish species that can provide new insights into the DNA damage response and embryo development. The transcriptomic and proteomic patterns changed significantly after exposure to genotoxicants in a stage-dependent manner. The results of this study indicate a correlation between phenotype formation and changes in transcriptomic and proteomic profiles. CPT and olaparib downregulated oxidative phosphorylation and metabolic pathways, and upregulated pathways involved in nucleotide excision repair, base excision repair, and homologous recombination. We observed the upregulated expression of zona pellucida sperm-binding proteins in all treatment groups, as well as the upregulation of several glycolytic enzymes. The analysis of gene expression revealed several markers of DNA damage response and adaptive stress response, which could be applied in toxicological studies on fish embryos. This study is the first complex analysis of the DNA damage response in endangered sturgeons.

The Lipid Composition of Euglena gracilis Middle Plastid Membrane Resembles That of Primary Plastid Envelopes.

Euglena gracilis is a photosynthetic flagellate possessing chlorophyte-derived secondary plastids that are enclosed by only three enveloping membranes, unlike most secondary plastids, which are surrounded by four membranes. It has generally been assumed that the two innermost E. gracilis plastid envelopes originated from the primary plastid, while the outermost is of eukaryotic origin. It was suggested that nucleus-encoded plastid proteins pass through the middle and innermost plastid envelopes of E. gracilis by machinery homologous to the translocons of outer and inner chloroplast membranes, respectively. Although recent genomic, transcriptomic, and proteomic data proved the presence of a reduced form of the translocon of inner membrane, they failed to identify any outer-membrane translocon homologs, which raised the question of the origin of E. gracilis's middle plastid envelope. Here, we compared the lipid composition of whole cells of the pigmented E. gracilis strain Z and two bleached mutants that lack detectable plastid structures, W10BSmL and WgmZOflL We determined the lipid composition of E. gracilis strain Z mitochondria and plastids, and of plastid subfractions (thylakoids and envelopes), using HPLC high-resolution tandem mass spectrometry, thin-layer chromatography, and gas chromatography-flame ionization detection analytical techniques. Phosphoglycerolipids are the main structural lipids in mitochondria, while glycosyldiacylglycerols are the major structural lipids of plastids and also predominate in extracts of whole mixotrophic cells. Glycosyldiacylglycerols were detected in both bleached mutants, indicating that mutant cells retain some plastid remnants. Additionally, we discuss the origin of the E. gracilis middle plastid envelope based on the lipid composition of envelope fraction.

The cell wall polysaccharides of a photosynthetic relative of apicomplexans, Chromera velia.

Chromerids are a group of alveolates, found in corals, that show peculiar morphological and genomic features. These organisms are evolutionary placed in-between symbiotic dinoflagellates and parasitic apicomplexans. There are two known species of chromerids: Chromera velia and Vitrella brassicaformis. Here, the biochemical composition of the C. velia cell wall was analyzed. Several polysaccharides adorn this structure, with glucose being the most abundant monosaccharide (approx. 80%) and predominantly 4-linked (approx. 60%), suggesting that the chromerids cell wall is mostly cellulosic. The presence of cellulose was cytochemically confirmed with calcofluor white staining of the algal cell. The remaining wall polysaccharides, assuming structures are similar to those of higher plants, are indicative of a mixture of galactans, xyloglucans, heteroxylans, and heteromannans. The present work provides, for the first time, insights into the outermost layers of the photosynthetic alveolate C. velia.

Isolation of plastids and mitochondria from Chromera velia.

MAIN CONCLUSION: We present an easy and effective procedure to purify plastids and mitochondria from Chromera velia. Our method enables downstream analyses of protein and metabolite content of the organelles. Chromerids are alveolate algae that are the closest known phototrophic relatives to apicomplexan parasites such as Plasmodium or Toxoplasma. While genomic and transcriptomic resources for chromerids are in place, tools and experimental conditions for proteomic studies have not been developed yet. Here we describe a rapid and efficient protocol for simultaneous isolation of plastids and mitochondria from the chromerid alga Chromera velia. This procedure involves enzymatic treatment and breakage of cells, followed by differential centrifugation. While plastids sediment in the first centrifugation step, mitochondria remain in the supernatant. Subsequently, plastids can be purified from the crude pellet by centrifugation on a discontinuous 60%/70% sucrose density gradient, while mitochondria can be obtained by centrifugation on a discontinuous 33%/80% Percoll density gradient. Isolated plastids are autofluorescent, and their multi-membrane structure was confirmed by transmission electron microscopy. Fluorescent optical microscopy was used to identify isolated mitochondria stained with MitoTrackerTM green, while their intactness and membrane potential were confirmed by staining with MitoTrackerTM orange CMTMRos. Total proteins were extracted from isolated organellar fractions, and the purity of isolated organelles was confirmed using immunoblotting. Antibodies against the beta subunit of the mitochondrial ATP synthase and the plastid protochlorophyllide oxidoreductase did not cross-react on immunoblots, suggesting that each organellar fraction is free of the residues of the other. The presented protocol represents an essential step for further proteomic, organellar, and cell biological studies of C. velia and can be employed, with minor optimizations, in other thick-walled unicellular algae.

Extracellular adenosine mediates a systemic metabolic switch during immune response.

Immune defense is energetically costly, and thus an effective response requires metabolic adaptation of the organism to reallocate energy from storage, growth, and development towards the immune system. We employ the natural infection of Drosophila with a parasitoid wasp to study energy regulation during immune response. To combat the invasion, the host must produce specialized immune cells (lamellocytes) that destroy the parasitoid egg. We show that a significant portion of nutrients are allocated to differentiating lamellocytes when they would otherwise be used for development. This systemic metabolic switch is mediated by extracellular adenosine released from immune cells. The switch is crucial for an effective immune response. Preventing adenosine transport from immune cells or blocking adenosine receptor precludes the metabolic switch and the deceleration of development, dramatically reducing host resistance. Adenosine thus serves as a signal that the "selfish" immune cells send during infection to secure more energy at the expense of other tissues.

Separation and identification of lipids in the photosynthetic cousins of Apicomplexa Chromera velia and Vitrella brassicaformis.

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.

Species Specificity of Aldehyde and Fatty Acid Profiles of Four Family Group Representatives within the Insect Infraorder Pentatomomorpha (Hemiptera: Heteroptera).

Reactive α,ß-unsaturated aldehydes, including 4-oxoalk-2-enals, are known to be present in volatile secretions of numerous heteropteran insect species. Because the aldehydes are likely to originate from metabolism of fatty acids (FAs), the present study aimed to examine and compare the aldehyde and FA profiles of four model heteropteran species. The model species consisted of adult family group representatives within the infraorder Pentatomomorpha (Hemiptera: Heteroptera): seed bug (Lygaeus equestris (Lygaeoidea)), dock leaf bug (Coreus marginatus (Coreoidea)), red firebug (Pyrrhocoris apterus (Pyrrhocoroidea)), and European stink bug (Graphosoma lineatum (Pentatomoidea)). Solid-phase microextraction combined with two-dimensional gas-chromatography/time-of-flight mass spectrometry was used to establish the profiles of volatile secretions in stressed living insects. The FA profiles of acylglyceride and phospholipid fractions deposited in fat body and/or hemolymph were obtained by liquid chromatography/mass spectrometry and gas chromatography with flame ionization detection techniques. Our results based on multivariate statistical analyses of the data imply that volatile secretion blends as well as fat body and/or hemolymph lipid profiles are species specific but the differences in volatile blends between different species do not mirror the changes in corresponding fat body and/or hemolymph lipid profiles of stressed and non-stressed individuals.

Divergent mitochondrial respiratory chains in phototrophic relatives of apicomplexan parasites.

Four respiratory complexes and ATP-synthase represent central functional units in mitochondria. In some mitochondria and derived anaerobic organelles, a few or all of these respiratory complexes have been lost during evolution. We show that the respiratory chain of Chromera velia, a phototrophic relative of parasitic apicomplexans, lacks complexes I and III, making it a uniquely reduced aerobic mitochondrion. In Chromera, putative lactate:cytochrome c oxidoreductases are predicted to transfer electrons from lactate to cytochrome c, rendering complex III unnecessary. The mitochondrial genome of Chromera has the smallest known protein-coding capacity of all mitochondria, encoding just cox1 and cox3 on heterogeneous linear molecules. In contrast, another photosynthetic relative of apicomplexans, Vitrella brassicaformis, retains the same set of genes as apicomplexans and dinoflagellates (cox1, cox3, and cob).

Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803.

The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.

Cost effective, robust, and reliable coupled separation techniques for the identification and quantification of phospholipids in complex biological matrices: application to insects.

The quantification of phospholipid classes and the determination of their molecular structures are crucial in physiological and medical studies. This paper's target analytes are cell membrane phospholipids, which play an important role in the seasonal acclimation processes of poikilothermic organisms. We introduce a set of simple and cost-effective analytical methods that enable efficient characterization and quantification of particular phospholipid classes and the identification and relative distribution of the individual phospholipid species. The analytical approach involves solid-phase extraction and high-performance thin-layer chromatography, which facilitate the separation of particular lipid classes. The obtained fractions are further transesterified to fatty acid methyl esters and subjected to gas chromatography coupled to flame ionization detection, which enables the determination of the position of double bonds. Phospholipid species separation is achieved by high-performance liquid chromatography with mass spectrometry, which gives information about the headgroup moiety and attached fatty acids. The total content of each phospholipids class is assessed by phosphorus determination by UV spectrophotometry. The simultaneous analysis of phosphorus, fatty acid residues, and phospholipid species provides detailed information about phospholipid composition. Evaluation of these coupled methods was achieved by application to an insect model, Pyrrhocoris apterus. High correlation was observed between fatty acid compositions as determined by gas chromatography and high-performance liquid chromatography analysis.

Melittin-The principal toxin of honeybee venom-Is also produced in the honeybee fat body.

Melittin is a powerful toxin present in honeybee venom that is active in a wide range of animals, from insects to humans. Melittin exerts numerous biological, toxicological, and pharmacological effects, the most important of which is destruction of the cell membrane. The phospholipase activity of melittin and its ability to activate phospholipases in the venom contribute to these actions. Using analytical methods, we discovered that the honeybee Apis mellifera produces melittin not only in the venom gland but also in its fat body cells, which remain resistant to this toxin's effects. We suggest that melittin acts as an anti-bacterial agent, since its gene expression is significantly upregulated when honeybees are infected with Escherichia coli and Listeria monocytogenes bacteria; additionally, melittin effectively kills these bacteria in the disc diffusion test. We hypothesize that the chemical and physicochemical properties of the melittin molecule (hydrophilicity, lipophilicity, and capacity to form tetramers) in combination with reactive conditions (melittin concentration, salt concentration, pH, and temperature) are responsible for the targeted destruction of bacterial cells and apparent tolerance towards own tissue cells. Considering that melittin is an important current and, importantly, potential broad-spectrum medication, a thorough understanding of the observed phenomena may significantly increase its use in clinical practice.

Runoff of foliar-applied natural fungicides in aquaponics: Implications for fish and nitrification.

Aquaponics is a method of producing food in a sustainable manner through the integration of aquaculture and hydroponics, which allows simultaneous cultivation of fish and economic crops. The use of natural fungicides are crucial to the sustainable control of diseases in aquaponics. We assessed the potential impacts of natural fungicides, such as clove oil and lecithin, as well as a synthetic fungicide, tebuconazole, following foliar application in aquaponics. This study examined the runoff rates of the fungicides in decoupled aquaponics, and the subsequent effects of the runoffs on nitrification processes and Nile tilapia (Oreochromis niloticus). The runoffs of the foliar-applied fungicides, clove oil, lecithin, and tebuconazole, were detected in aquaponics water at a percentage runoff rate of 0.3 %, 2.3 %, and 0.3-0.8 % respectively. In the biofilter, lecithin altered the ammonium levels by increasing ammonium-nitrogen levels by 7 mg L-1, 6 h post application. Clove oil, on the other hand, showed no significant effect on ammonium, nitrite, and nitrate-nitrogen. Similarly, the toxicity test showed that eugenol had no significant effects on the hematological, biochemical and antioxidative activities of O. niloticus. Conversely, tebuconazole exhibited significant and persistent effects on various biochemical parameters, including lactate, albumin, and total protein, as well as hematological parameters like hemoglobin and MCH. The use of lecithin and tebuconazole should only be limited to decoupled aquaponics.

Circadian rhythms and circadian clock gene homologs of complex alga Chromera velia.

Most organisms on Earth are affected by periodic changes in their environment. The circadian clock is an endogenous device that synchronizes behavior, physiology, or biochemical processes to an approximately 24-hour cycle, allowing organisms to anticipate the periodic changes of day and night. Although circadian clocks are widespread in organisms, the actual molecular components differ remarkably among the clocks of plants, animals, fungi, and prokaryotes. Chromera velia is the closest known photosynthetic relative of apicomplexan parasites. Formation of its motile stage, zoospores, has been described as associated with the light part of the day. We examined the effects on the periodic release of the zoospores under different light conditions and investigated the influence of the spectral composition on zoosporogenesis. We performed a genomic search for homologs of known circadian clock genes. Our results demonstrate the presence of an almost 24-hour free-running cycle of zoosporogenesis. We also identified the blue light spectra as the essential compound for zoosporogenesis. Further, we developed a new and effective method for zoospore separation from the culture and estimated the average motility speed and lifespan of the C. velia zoospores. Our genomic search identified six cryptochrome-like genes, two genes possibly related to Arabidopsis thaliana CCA/LHY, whereas no homolog of an animal, cyanobacterial, or fungal circadian clock gene was found. Our results suggest that C. velia has a functional circadian clock, probably based mainly on a yet undefined mechanism.

Stylopsal: the first identified female-produced sex pheromone of strepsiptera.

A female-produced sex pheromone of Stylops muelleri was identified as an unusually branched saturated aldehyde (9R)-3,5-syn-3,5,9-trimethyldodecanal. We named it stylopsal. Its structure was established by using mass spectrometry, infrared spectroscopy, and organic synthesis of candidate compounds. The synthetic standard of (9R)-3,5-syn-3,5,9-trimethyldodecanal gave identical chromatographic and mass spectrometric data as the natural pheromone and also was active in electroantennographic and behavioral assays. The female fat body lipids contained the corresponding fatty acid, indicating a possible link between lipid metabolism and the pheromone biosynthesis.

Fatty Acid Biosynthesis in Chromerids.

Fatty acids are essential components of biological membranes, important for the maintenance of cellular structures, especially in organisms with complex life cycles like protozoan parasites. Apicomplexans are obligate parasites responsible for various deadly diseases of humans and livestock. We analyzed the fatty acids produced by the closest phototrophic relatives of parasitic apicomplexans, the chromerids Chromera velia and Vitrella brassicaformis, and investigated the genes coding for enzymes involved in fatty acids biosynthesis in chromerids, in comparison to their parasitic relatives. Based on evidence from genomic and metabolomic data, we propose a model of fatty acid synthesis in chromerids: the plastid-localized FAS-II pathway is responsible for the de novo synthesis of fatty acids reaching the maximum length of 18 carbon units. Short saturated fatty acids (C14:0-C18:0) originate from the plastid are then elongated and desaturated in the cytosol and the endoplasmic reticulum. We identified giant FAS I-like multi-modular enzymes in both chromerids, which seem to be involved in polyketide synthesis and fatty acid elongation. This full-scale description of the biosynthesis of fatty acids and their derivatives provides important insights into the reductive evolutionary transition of a phototropic algal ancestor to obligate parasites.

The Cryptic Plastid of Euglena longa Defines a New Type of Nonphotosynthetic Plastid Organelle.

Most secondary nonphotosynthetic eukaryotes have retained residual plastids whose physiological role is often still unknown. One such example is Euglena longa, a close nonphotosynthetic relative of Euglena gracilis harboring a plastid organelle of enigmatic function. By mining transcriptome data from E. longa, we finally provide an overview of metabolic processes localized to its elusive plastid. The organelle plays no role in the biosynthesis of isoprenoid precursors and fatty acids and has a very limited repertoire of pathways concerning nitrogen-containing metabolites. In contrast, the synthesis of phospholipids and glycolipids has been preserved, curiously with the last step of sulfoquinovosyldiacylglycerol synthesis being catalyzed by the SqdX form of an enzyme so far known only from bacteria. Notably, we show that the E. longa plastid synthesizes tocopherols and a phylloquinone derivative, the first such report for nonphotosynthetic plastids studied so far. The most striking attribute of the organelle could be the presence of a linearized Calvin-Benson (CB) pathway, including RuBisCO yet lacking the gluconeogenetic part of the standard cycle, together with ferredoxin-NADP+ reductase (FNR) and the ferredoxin/thioredoxin system. We hypothesize that the ferredoxin/thioredoxin system activates the linear CB pathway in response to the redox status of the E. longa cell and speculate on the role of the pathway in keeping the redox balance of the cell. Altogether, the E. longa plastid defines a new class of relic plastids that is drastically different from the best-studied organelle of this category, the apicoplast.IMPORTANCE Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa, a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO2-fixing enzyme RuBisCO in E. longa Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids.

First Comprehensive Study of a Giant Among the Insects, Titanus giganteus: Basic Facts from Its Biochemistry, Physiology, and Anatomy.

Titanus giganteus is one of the largest insects in the world, but unfortunately, there is a lack of basic information about its biology. Previous papers have mostly described Titanus morphology or taxonomy, but studies concerning its anatomy and physiology are largely absent. Thus, we employed microscopic, physiological, and analytical methods to partially fill this gap. Our study focused on a detailed analysis of the antennal sensilla, where coeloconic sensilla, grouped into irregularly oval fields, and sensilla trichoidea were found. Further, the inspection of the internal organs showed apparent degeneration of the gut and almost total absence of fat body. The gut was already empty; however, certain activity of digestive enzymes was recorded. The brain was relatively small, and the ventral nerve cord consisted of three ganglia in the thorax and four ganglia in the abdomen. Each testis was composed of approximately 30 testicular follicles filled with a clearly visible sperm. Chromatographic analysis of lipids in the flight muscles showed the prevalence of storage lipids that contained 13 fatty acids, and oleic acid represented 60% of them. Some of our findings indicate that adult Titanus rely on previously accumulated reserves rather than feeding from the time of eclosion.

Liver macrophages regulate systemic metabolism through non-inflammatory factors.

Liver macrophages (LMs) have been proposed to contribute to metabolic disease through secretion of inflammatory cytokines. However, anti-inflammatory drugs lead to only modest improvements in systemic metabolism. Here we show that LMs do not undergo a proinflammatory phenotypic switch in obesity-induced insulin resistance in flies, mice and humans. Instead, we find that LMs produce non-inflammatory factors, such as insulin-like growth factor-binding protein 7 (IGFBP7), that directly regulate liver metabolism. IGFBP7 binds to the insulin receptor and induces lipogenesis and gluconeogenesis via activation of extracellular-signal-regulated kinase (ERK) signalling. We further show that IGFBP7 is subject to RNA editing at a higher frequency in insulin-resistant than in insulin-sensitive obese patients (90% versus 30%, respectively), resulting in an IGFBP7 isoform with potentially higher capacity to bind to the insulin receptor. Our study demonstrates that LMs can contribute to insulin resistance independently of their inflammatory status and indicates that non-inflammatory factors produced by macrophages might represent new drug targets for the treatment of metabolic diseases.

Author Correction: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

In the version of this article initially published, author Volker M. Lauschke had affiliation number 13; the correct affiliation number is 12. The error has been corrected in the HTML and PDF versions of the article.

Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster.

Adaptative responses of ectothermic organisms to thermal variation typically involve the reorganization of membrane glycerophospholipids (GPLs) to maintain membrane function. We investigated how acclimation at 15, 20 and 25 degrees C during preimaginal development influences the thermal tolerance and the composition of membrane GPLs in adult Drosophila melanogaster. Long-term cold survival was significantly improved by low acclimation temperature. After 60 h at 0 degrees C, more than 80% of the 15 degrees C-acclimated flies survived while none of the 25 degrees C-acclimated flies survived. Cold shock tolerance (1h at subzero temperatures) was also slightly better in the cold acclimated flies. LT50 shifted down by ca 1.5 degrees C in 15 degrees C-acclimated flies in comparison to those acclimated at 25 degrees C. In contrast, heat tolerance was not influenced by acclimation temperature. Low temperature acclimation was associated with the increase in proportion of ethanolamine (from 52.7% to 58.5% in 25 degrees C-acclimated versus 15 degrees C-acclimated flies, respectively) at the expense of choline in GPLs. Relatively small, but statistically significant changes in lipid molecular composition were observed with decreasing acclimation temperature. In particular, the proportions of glycerophosphoethanolamines with linoleic acid (18:2) at the sn-2 position increased. No overall change in the degree of fatty acid unsaturation was observed. Thus, cold tolerance but not heat tolerance was influenced by preimaginal acclimation temperature and correlated with the changes in GPL composition in membranes of adult D. melanogaster.