Antiaphrodisiacs are chemical bouquets physically delivered from male to female individuals upon copulation which discourage further mating and reduce sperm competition by rendering the female less attractive. Since antiaphrodisiacs may not offer an honest signal of female receptivity, in polyandrous species they may undergo faster diversification resulting from sexual conflict. The Heliconiini tribe of butterflies includes a polyandrous (free-mating) and a monandrous (pupal-mating) clade, both known to produce diverse antiaphrodisiac mixtures as part of their abdominal blends. Using multivariate phylogenetic comparative methods, we analyzed the genital blends of 36 Heliconiini species to test the hypothesis that blend diversity results from male-male competition in polyandry. We found no evidence for shifts in blend diversification rate corresponding to changes in mating strategy, implying male-male competition may have a weaker effect on pheromone diversification in this group than previously thought. The genital blends of most species are dominated by one of four highly volatile compounds; (E)-β-ocimene, octen-3-one, sulcatone and 4-hydroxycyclopent-2-en-1-one. Based on the function of (E)-β-ocimene as the behaviourally active antiaphrodisiac in H. melpomene, we propose a similar role in other species for the other volatiles. We test this hypothesis by investigating 4-hydroxycyclopent-2-en-1-one occurrence in Heliconius sara. While we detect no sex-based differences on its presence, we find the compound is undetectable when larvae are not fed their preferred host plant, providing an intriguing potential link between host plant and reproductive cues. This in turn shows that captive-bred samples do not always provide realistic results and this awareness is important for future experiments.

Organic residue analysis (ORA) is a valuable tool for the study of ancient diets, but conventional methods remain limited in terms of taxonomic identification or to resolve mixtures. Here, we propose a method to further explore a class of compounds-triacylglycerols (TAGs)-using high-resolution mass spectrometry to overcome these limitations in an attempt to better characterise culinary practices. Over 70 medieval Sicilian pots and a wide range of authentic fresh products were studied by matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS and MALDI-MS/MS). MALDI-MS analysis can distinguish fresh foodstuffs but provides little additional information regarding the contents of archaeological pottery compared to conventional ORA methods. In contrast, product ion analyses were able to deconvolute a range of animal carcass fat mixtures. In addition, detailed analysis of the composition of saturated T44 and unsaturated T50-T54 TAGs was able to provide greater taxonomic resolution regarding dairy products and plant oils.

Sphingolipids are ubiquitous in membranes of eukaryotes and are associated with important cellular functions. Although sphingolipids occur scarcely in bacteria, for some of them they are essential and, in other bacteria, they contribute to fitness and stability of the outer membrane, such as in the well-studied α-proteobacterium Caulobacter crescentus. We previously defined five structural genes for ceramide synthesis in C. crescentus, among them the gene for serine palmitoyltransferase, the enzyme that catalyzes the committed step of sphingolipid biosynthesis. Other mutants affected in genes of this same genomic region show cofitness with a mutant deficient in serine palmitoyltransferase. Here we show that at least two phosphosphingolipids are produced in C. crescentus and that at least another six gene products are needed for the decoration of ceramide upon phosphosphingolipid formation. All eleven genes participating in phosphosphingolipid formation are also required in C. crescentus for membrane stability and for displaying sensitivity towards the antibiotic polymyxin B. The genes for the formation of complex phosphosphingolipids are also required for C. crescentus virulence on Galleria mellonella insect larvae.

Sphingolipids are ubiquitous in membranes of eukaryotes and are associated with important cellular functions. Although sphingolipids occur scarcely in bacteria, for some of them they are essential and, in other bacteria, they contribute to fitness and stability of the outer membrane, such as in the well-studied α-proteobacterium Caulobacter crescentus . We previously defined five structural genes for ceramide synthesis in C. crescentus . However, other mutants affected in genes of this same genomic region show cofitness with a mutant deficient in serine palmitoyltransferase. Here we show that at least two phospho-sphingolipids are produced in C. crescentus and that at least another six gene products are needed for the decoration of ceramide upon phospho-sphingolipid formation. All eleven genes participating in phospho-sphingolipid formation are also required in C. crescentus for membrane stability and for displaying sensitivity towards the antibiotic polymyxin B. The genes for the formation of complex phospho-sphingolipids are also required for C. crescentus virulence on Galleria mellonella insect larvae.

Pheromone communication is widespread among animals. Since it is often involved in mate choice, pheromone production is often tightly controlled. Although male sex pheromones (MSPs) and anti-aphrodisiacs have been studied in some Heliconius butterfly species, little is known about the factors affecting their production and release in these long-lived butterflies. Here, we investigate the effect of post-eclosion age on chemical blends from pheromone-emitting tissues in Heliconius atthis and Heliconius charithonia, exhibiting respectively free-mating and pupal-mating strategies that are hypothesised to differently affect the timing of their pheromone emissions. We focus on two different tissues: the wing androconia, responsible for MSPs used in courtship, and the genital tip, the production site for anti-aphrodisiac pheromones that affect post-mating behaviour. Gas chromatography-mass spectrometric analysis of tissue extracts from virgin males and females of both species from day 0 to 8 post-eclosion demonstrates the following. Some ubiquitous fatty acid precursors are already detectable at day 0. The complexity of the chemical blends increases with age regardless of tissue or sex. No obvious difference in the time course of blend production was evident between the two species, but female tissues in H. charithonia were more affected by age than in H. atthis. We suggest that compounds unique to male androconia and genitals and whose amount increases with age are potential candidates for future investigation into their roles as pheromones. While this analysis revealed some of the complexity in Heliconius chemical ecology, the effects of other factors, such as the time of day, remain unknown.

The recovery of visually recognizable brain masses from skeletonized human burials is rare; the discovery of an exceptionally well-preserved 2500-year-old brain during excavations at Heslington, Yorkshire, UK, therefore attracted international publicity. Analysis of a lipidic extract of that brain material showed the presence of high molecular weight organic components. To determine whether the presence of such compounds is a common feature of unusually preserved brain specimens, we have carried out high resolution matrix-assisted laser desorption/ionization Fourier transform ion-cyclotron resonance mass spectrometric (MALDI FT-ICR-MS) analyses of lipidic extracts of preserved brain remains from 14 contexts, obtained from four archaeological sites ranging from 200 to 2500 years old. These data allowed elemental formulae for the predominant signals in the resulting spectra to be generated, and a detailed comparison of the resulting very rich dataset was carried out, allowing 38 molecular formulae to be assigned. The components identified across all the sample extracts analysed fell into the same class of large organic molecules as those from the Heslington brain and are generally functionalized with low numbers of heteroatoms (oxygen, nitrogen, or both). These components do not correspond to those identified in adipocere, but instead resemble kerogen substructures, surprisingly formed over historic rather than geological timescales. The presence of such insoluble, inert structures would help explain why brain material in which they are found is persistent. Our results pave the way for further analyses aimed at understanding the processes that take place when brain material is preserved in the archaeological record.

During courtship, male butterflies of many species produce androconial secretions containing male sex pheromones (MSPs) that communicate species identity and affect female choice. MSPs are thus likely candidates as reproductive barriers, yet their role in speciation remains poorly studied. Although Heliconius butterflies are a model system in speciation, their MSPs have not been investigated from a macroevolutionary perspective. We use GC/MS to characterize male androconial secretions in 33 of the 69 species in the Heliconiini tribe. We found these blends to be species-specific, consistent with a role in reproductive isolation. We detected a burst in blend diversification rate at the most speciose genus, Heliconius ; a consequence of Heliconius and Eueides species using a fatty acid (FA) metabolic pathway to unlock more complex blends than basal Heliconiini species, whose secretions are dominated by plant-like metabolites. A comparison of 10 sister species pairs demonstrates a striking positive correlation between blend dissimilarity and range overlap, consistent with character displacement or reinforcement in sympatry. These results demonstrate for the first time that MSP diversification can promote reproductive isolation across this group of butterflies, showcasing how implementation of an ancestral trait, the co-option of the FA metabolic pathway for pheromone production, can facilitate rapid speciation.

ABSTRACT Sulfoquinovosyl diacylglycerol (SQDG) is a membrane-forming lipid present in photosynthetic organisms as well as in distinct bacteria growing in phosphate-limited environments. Four genes for SQDG biosynthesis were previously identified in Rhodobacter sphaeroides , the operon sqdBDC and sqdA . In this work, we found that SMc02490 of Sinorhizobium meliloti is an SqdA orthologue. Expression of the S. meliloti sqdBDC operon in Escherichia coli results in formation of sulfoquinovosyl glycerol (SQGro), while co-expression of this operon together with smc02490 ( sqdA) results in formation of SQDG. Furthermore, SqdA allows for incorporation of exogenous SQGro into SQDG in S. meliloti and in E. coli cultures, suggesting that presence of SqdA in bacteria permit them to use environmental SQGro for the biosynthesis of the membrane lipid SQDG. An in vitro enzymatic assay for the acyltransferase SqdA was developed. Cell-free crude extracts of E. coli expressing sqdA can efficiently convert [ 35 S]-sulfoquinovosyl monoacylglycerol into SQDG using as acyl donor acyl carrier protein. Bioinformatic analyses reveal that this sulfoquinovose acylation pathway for SQDG biosynthesis is delimited to the Hyphomicrobiales (Rhizobiales) and Rhodobacterales orders of Alphaproteobacteria.

This study aimed to investigate the highly differentiated urothelial apical surface glycome. The functions of the mammalian urothelium, lining the majority of the urinary tract and providing a barrier against toxins in urine, are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification, and complex assembly to regulate the formation of multiple differentiated cell layers. Protein glycosylation, a poorly studied aspect of urothelial differentiation, contributes to the apical glycome and is implicated in the development of urothelial diseases. To enable surface glycome characterization, we developed a method to collect tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies was developed for enzymatic shaving of the luminal bladder urothelial surface, with subsequent release and mass spectrometric analysis of apical surface O- and N-glycans, the first normal mammalian urothelial N-glycome to be defined. Trypsinization of superficial glycoproteins was tracked using immunolabeling of the apically expressed uroplakin 3a protein to optimize enzymatic release, without compromising the integrity of the superficial urothelial layer. The approach developed for releasing apical tissue surface glycans allowed for comparison with the N-glycome of the total porcine bladder urothelial cells and thus identification of apical surface glycans as candidates implicated in the urothelial barrier function. Data are available in MassIve: MSV000087851.

Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is widely used for the analysis of biomolecules. Label-assisted laser desorption/ionisation mass spectrometry (LALDI-MS) is a matrix-free variant of MALDI-MS, in which only analytes covalently attached to a laser desorption/ionisation (LDI) enhancer are detected. LALDI-MS has shown promise in overcoming the limitations of MALDI-MS in terms of sample preparation and MS analysis. In this work, we have developed a series of pyrene-based LDI reagents (LALDI tags) that can be used for labelling and LALDI-MS analysis of reducing carbohydrates from complex (biological) samples without the need for additional chemical derivatisation or purification. We have systematically explored the suitability of four pyrene-based LDI enhancers and three aldehyde-reactive handles, optimised sample preparation, and demonstrated the use of LALDI tags for the detection of lactose. We have also exemplified the potential of LALDI tags for labelling carbohydrates in biological samples by direct detection of lactose in cow's milk. These results demonstrate that LALDI-MS is a promising technique for the analysis of reducing carbohydrates in biological samples, and pave the way for the development of LALDI-MS for glycomics and diagnostics.

Fast Atom Bombardment (FAB) is a mass spectrometric (MS) ionization technique which is particularly suited for the examination of biomolecules such as carbohydrates. Fast Atom Bombardment (FAB) is a mass spectrometric (MS) ionization technique which is particularly suited for the examination of biomolecules such as carbohydrates. The individual signals in a cluster will be observed when the instrumental resolution is set to resolve nominal masses. A good example of where a modification constitutes an improvement over traditional protocols is the peracetylation reaction. Permethylation techniques are ubiquitous in carbohydrate analysis and are almost always based on Hakomori's classical method with a few notable exceptions. The way in which a sample is analyzed depends on the nature of the compound, how it has been prepared, and what sort of data is required from it. The abundance of fragment ions can sometimes be enhanced by the use of collision gases.

Metabolomics is a technique that allows for the evaluation of the entire extractable chemical profile of a plant, for example, using high-resolution mass spectrometry (HRMS) and can be used to evaluate plant stress responses, such as those due to drought. Metabolomic analysis is dependent upon the efficiency of the extraction protocol. Currently, there are two common extraction procedures widely used in metabolomic experiments, those that extract from plant tissue processed in liquid nitrogen or extraction from lyophilised plant tissues. Here, we evaluated the two using non-targeted metabolomics to show that lyophilisation can stabilise the maize (Zea mays) extractable metabolome, increasing throughput and efficiency of extraction as compared to the more traditional processing in liquid nitrogen. Then, we applied the lyophilisation approach to explore the effect of drought upon the maize metabolome in a non-targeted HRMS metabolomics approach. Metabolomics revealed differences in the mature maize metabolome having undergone three drought conditions imposed at two critical development stages (three-leaf stage and grain-fill stage); moreover, this difference was observed across two tissue types (kernel and inner cob/pith). It was shown that under ideal conditions, the biochemical make-up of the tissue types is different. However, under stress conditions, the stress response dominates the metabolic profile. Drought-related metabolites known from other plant systems have been identified and metabolomics has revealed potential novel drought-stress indicators in our maize system.

The pinewood nematode (PWN) is the causal agent of pine wilt disease, a pathology that affects conifer forests, mainly Pinus spp. PWN infection can induce the expression of phytohormone-related genes; however, changes at the early phytohormone level have not yet been explored. Phytohormones are low-abundance metabolites, and thus, difficult to quantify. Moreover, most methodologies focus mainly on Arabidopsis or crop species. This work aimed to validate a fast (run time 6.6 min) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) analytical method to quantify 14 phytohormones in Pinus pinaster stem tissues. This method was further applied to evaluate, for the first time, early phytohormone changes in susceptible and resistant phenotypes of P. pinaster 24, 48 and 72 h after inoculation (HAI) with PWN. A significant increase in salicylic acid (SA, 48 and 72 HAI) and jasmonic acid methyl ester (JA-ME, 72 HAI) was observed in susceptible phenotypes. Results indicate that the higher susceptibility of P. pinaster to PWN infection might result from an inefficient trigger of hypersensitive responses, with the involvement of JA and SA pathways. This work provides an important update in forest research, and adds to the current knowledge of Pinus spp. defence responses to PWN infection.

Brain tissue is ubiquitous in the archaeological record. Multiple, independent studies report the finding of black, resinous or shiny brain tissue, and Petrone et al. [2020 “Heat-induced Brain Vitrification from the Vesuvius Eruption in C.E. 79.” N Engl J Med. 382: 383–384; doi:10.1056/NEJMc1909867] raise the intriguing prospect of a role for vitrification in the preservation of ancient biomolecules. However, Petrone et al. (2020) have not made their raw data available, and no detailed laboratory or analytical methodology is offered. Issues of contamination and misinterpretation hampered a decade of research in biomolecular archaeology, such that addressing these sources of bias and facilitating validation of specious findings has become both routine and of paramount importance in the discipline. We argue that the evidence they present does not support their conclusion of heat-induced vitrification of human brain tissue, and that future studies should share palaeoproteomic data in an open access repository to facilitate comparative analysis of the recovery of ancient proteins and patterns of their degradation.

Chemical analysis of archaeological artefacts is used with increasing regularity to understand how wine was produced, traded, and consumed in the past and to shed light on its antiquity. Based both on an extensive review of the published literature and on new analyses, here we critically evaluate the diverse range of methodological approaches that have been used for wine identification. Overall, we conclude that currently none of the proposed chemical 'biomarkers' for wine provide unequivocal evidence. Nevertheless, valid interpretations may be offered if systematically supported by additional contextual data, such as archaeobotanical evidence. We found the extraction and detection method to be particularly crucial for successful identification. We urge the use of controls and quantification to rule out false positives. DNA sequencing offers potential for identifying wine and provides much higher taxonomic resolution, but work is needed to determine the limits of DNA survival on artefacts.

Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is widely used for the analysis of biomolecules. Label-assisted laser desorption/ionisation mass spectrometry (LALDI-MS) is a matrix-free variant of MALDI-MS, in which only analytes covalently attached to a laser desorption/ionisation (LDI) enhancer are detected. LALDI-MS has shown promise in overcoming limitations of MALDI-MS in terms of sample preparation and MS analysis. In this work, we have developed water-soluble pyrene-based LDI reagents (LALDI tags) that can be used for (in situ) labelling and LALDI-MS analysis of reducing carbohydrates from complex (biological) samples without the need for additional chemical derivatisation or purification. We have systematically explored the suitability of four pyrene-based LDI enhancers and three aldehyde-reactive handles, optimised sample preparation, and exemplified the use of a LALDI tag by the detection of lactose in cow’s milk. These results demonstrate that LALDI-MS is a promising technique for the analysis of reducing carbohydrates in biological samples, and pave the way for the development of LALDI-MS for glycomics and diagnostics.

SummarySphingolipids are essential and common membrane components in eukaryotic organisms, participating in many important cellular functions. Only a few bacteria are thought to harbour sphingolipids in their membranes, among them the well‐studied α‐proteobacterium Caulobacter crescentus, a model organism for asymmetric cell division and cellular differentiation. Here, we report that C. crescentus wild type produces several molecular species of dihydroceramides, which are not produced in a mutant lacking the structural gene for serine palmitoyltransferase (spt). Whereas growth of a spt‐deficient mutant and wild type are indistinguishable during the exponential phase of growth, survival of the spt‐deficient mutant is much reduced, in comparison with wild type, during stationary phase of growth, especially at elevated temperatures. The structural gene for spt is located within a genomic cluster, comprising another 16 genes and which, like spt, are important for fitness of C. crescentus. Mutants deficient in genes linked to spt by high cofitness were unable to produce dihydroceramide or to survive in stationary phase of growth at elevated temperatures. At least five structural genes are required for dihydroceramide biosynthesis in C. crescentus and sphingolipid biosynthesis is needed for survival of this bacterium and the integrity of its outer membrane.