Trail pheromone identification in the ant Crematogaster scutellaris.

In this work, we identified the trail pheromone of the ant Crematogaster scutellaris. We combined gas chromatography-mass spectrometry analysis of extracts from the hind tibia, the location of the respective glands, with automated trail following assays. The study found tridecan-2-ol to be the strongest discriminator between hind tibia and other body part extracts. Tridecan-2-ol elicited trail-following behaviour at concentrations of 1 ng/µL. A separation of the enantiomers showed responses to (R)-tridecan-2-ol already at 0.001 ng/µL and only at a 1000-fold higher concentration for (S)-tridecan-2-ol, suggesting that only the R enantiomer is used by C. scutellaris in its natural environment. We also found strong behavioural responses to 2-dodecanol, a substance that was not detectable in the hind tibia extract of C. scutellaris, but which has been reported to be the trail pheromone of the related species C. castanea. We discuss the contribution of these results to the 'dissection and reconstruction' of strategies and mechanisms underlying the social organization of ants.

Changes in phospholipid profiles in early larval stages of the marine mussel Mytilus galloprovincialis indicate a role of ceramides in bivalve development.

BACKGROUND: Phospholipids are highly diverse molecules with pleiotropic biological roles, from membrane components and signaling molecules, whose composition can change in response to both endogenous and external stimuli. Recent lipidomic studies on edible bivalve mollusks were focused on lipid nutritional value and growth requirements. However, no data are available on phospholipid profiles during bivalve larval development. In the model marine bivalve Mytilus galloprovincialis, early larvae (up to 48 hours post fertilization-hpf) undergo dramatic molecular and functional changes, including shell biogenesis and neurogenesis, that are sustained by egg lipid reserves. Changes in phospholipid composition may also occur participating in the complex processes of early development. OBJECTIVE: The lipidome of M. galloprovincialis eggs and early larval stages (24 and 48 hpf) was investigated in order to identify possible changes in phospholipid classes and related metabolic pathways that may play a role in key steps of development. MATERIALS AND METHODS: Lipidomic analysis were performed by NMR spectroscopy and liquid chromatography-mass spectrometry (LC-MS), with focus on phospholipids. Shifts in relative species composition of phosphatidylcholine, phosphatidylethanolamine, plasmalogen, and ceramide aminoethylphosphonate-CAEP, the bivalve analogue of the main mammalian ceramide sphingomyelin, were observed. Expression of genes involved in ceramide homeostasis was also modulated from eggs to early larval stages. RESULTS: The results represent the first data on changes in phospholipid composition in bivalve larvae and suggest a functional role of phospholipids in mussel early development. CONCLUSION: The results underline the importance of lipidomic studies in bivalve larvae, in both physiological conditions and in response to environmental stress.

Assessing the toxicity of aegerolysin-based bioinsecticidal complexes using the sea urchin Paracentrotus lividus as model organism.

The use of alternative solutions for pest management to replace pesticides in agriculture is of great interest. Proteinaceous complexes deriving from edible oyster mushrooms were recently proposed as environmentally friendly bioinsecticides. Such complexes, composed of ostreolysin A6 (OlyA6) and pleurotolysin B (PlyB), target invertebrate-specific membrane sphingolipids in insect's midgut, causing death through the formation of transmembrane pores. In this work, the potential impact of OlyA6/PlyB complexes was tested in the Mediterranean sea urchin Paracentrotus lividus, as an indicator of environmental quality. The ability of the fluorescently tagged OlyA6 to bind sea urchin gametes (sperm, eggs), the lipidome of sea urchin gametes, and the potential toxic effects and developmental anomalies caused by OlyA6/PlyB complexes on P. lividus early development (embryo, larvae) were investigated. The binding of the fluorescently tagged OlyA6 could be observed only in sea urchin eggs, which harbor OlyA6 sphingolipid membrane receptors, conversely to sperm. High protein concentrations affected sea urchin fertilization (>750 µg/L) and early development (> 375 µg/L in embryos; >100 µg/L in larvae), by causing toxicity and morphological anomalies in embryos and larvae. The main anomalies consisted in delayed embryos and incorrect migration of the primary mesenchyme cells that caused larval skeletal anomalies. The classification of these anomalies indicated a slight environmental impact of OlyA6/PlyB complexes at concentrations higher than 750 µg/L. Such impact should not persist in the marine environment, due to the reversible anomalies observed in sea urchin embryos and larvae that may promote defense strategies. However, before promoting the use of OlyA6/PlyB complexes as bio-pesticides at low concentrations, further studies on other marine coastal species are needed.

Chemical Defence by Sterols in the Freshwater Ciliate Stentor polymorphus.

Heterotrich ciliates typically retain toxic substances in specialized ejectable organelles, called extrusomes, which are used in predator-prey interactions. In this study, we analysed the chemical defence strategy of the freshwater heterotrich ciliate Stentor polymorphus against the predatory ciliate Coleps hirtus, and the microturbellarian flatworm Stenostomum sphagnetorum. The results showed that S. polymorphus is able to defend itself against these two predators by deploying a mix of bioactive sterols contained in its extrusomes. Sterols were isolated in vivo and characterized by liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR), as ergosterol, 7-dehydroporiferasterol, and their two peroxidized analogues. The assessment of the toxicity of ergosterol and ergosterol peroxide against various organisms, indicated that these sterols are essential for the effectiveness of the chemical defence in S. polymorphus.

Expansion and Neofunctionalization of Actinoporin-like Genes in Mediterranean Mussel (Mytilus galloprovincialis).

Pore-forming toxins are an important component of the venom of many animals. Actinoporins are potent cytolysins that were first detected in the venom of sea anemones; however, they are occasionally found in animals other than cnidarians and are expanded in a few predatory gastropods. Here, we report the presence of 27 unique actinoporin-like genes with monophyletic origin in Mytilus galloprovincialis, which we have termed mytiporins. These mytiporins exhibited a remarkable level of molecular diversity and gene presence-absence variation, which warranted further studies aimed at elucidating their functional role. We structurally and functionally characterized mytiporin-1 and found significant differences from the archetypal actinoporin fragaceatoxin C. Mytiporin-1 showed weaker permeabilization activity, no specificity towards sphingomyelin, and weak activity in model lipid systems with negatively charged lipids. In contrast to fragaceatoxin C, which forms octameric pores, functional mytiporin-1 pores on negatively charged lipid membranes were hexameric. Similar hexameric pores were observed for coluporin-26 from Cumia reticulata and a conoporin from Conus andremenezi. This indicates that also other molluscan actinoporin-like proteins differ from fragaceatoxin C. Although the functional role of mytiporins in the context of molluscan physiology remains to be elucidated, the lineage-specific gene family expansion event that characterizes mytiporins indicates that strong selective forces acted on their molecular diversification. Given the tissue distribution of mytiporins, this process may have broadened the taxonomic breadth of their biological targets, which would have important implications for digestive processes or mucosal immunity.

Functional Study of Lipoxygenase-Mediated Resistance against Fusarium verticillioides and Aspergillus flavus Infection in Maize.

Mycotoxin contamination of maize kernels by fungal pathogens like Fusarium verticillioides and Aspergillus flavus is a chronic global challenge impacting food and feed security, health, and trade. Maize lipoxygenase genes (ZmLOXs) synthetize oxylipins that play defense roles and govern host-fungal interactions. The current study investigated the involvement of ZmLOXs in maize resistance against these two fungi. A considerable intraspecific genetic and transcript variability of the ZmLOX family was highlighted by in silico analysis comparing publicly available maize pan-genomes and pan-transcriptomes, respectively. Then, phenotyping and expression analysis of ZmLOX genes along with key genes involved in oxylipin biosynthesis were carried out in a maize mutant carrying a Mu transposon insertion in the ZmLOX4 gene (named UFMulox4) together with Tzi18, Mo17, and W22 inbred lines at 3- and 7-days post-inoculation with F. verticillioides and A. flavus. Tzi18 showed the highest resistance to the pathogens coupled with the lowest mycotoxin accumulation, while UFMulox4 was highly susceptible to both pathogens with the most elevated mycotoxin content. F. verticillioides inoculation determined a stronger induction of ZmLOXs and maize allene oxide synthase genes as compared to A. flavus. Additionally, oxylipin analysis revealed prevalent linoleic (18:2) peroxidation by 9-LOXs, the accumulation of 10-oxo-11-phytoenoic acid (10-OPEA), and triglyceride peroxidation only in F. verticillioides inoculated kernels of resistant genotypes.

Valorization of Traditional Italian Walnut (Juglans regia L.) Production: Genetic, Nutritional and Sensory Characterization of Locally Grown Varieties in the Trentino Region.

Juglans regia (L.) is cultivated worldwide for its nutrient-rich nuts. In Italy, despite the growing demand, walnut cultivation has gone through a strong decline in recent decades, which led to Italy being among the top five net importing countries. To promote the development of local high-quality Italian walnut production, we devised a multidisciplinary project to highlight the distinctive traits of three varieties grown in the mountainous region Trentino (northeast of Italy): the heirloom 'Bleggiana', a second local accession called local Franquette and the French cultivar 'Lara', recently introduced in the local production to increase yield. The genetic characterization confirmed the uniqueness of 'Bleggiana' and revealed local Franquette as a newly described autochthonous variety, thus named 'Blegette'. The metabolic profiles highlighted a valuable nutritional composition of the local varieties, richer in polyphenols and with a lower ω-6/ω-3 ratio than the commercial 'Lara'. 'Blegette' obtained the highest preference scores from consumers for both the visual aspect and tasting; however, the volatile organic compound profiles did not discriminate among the characterized cultivars. The described local varieties represent an interesting reservoir of walnut genetic diversity and quality properties, which deserve future investigation on agronomically useful traits (e.g., local adaptation and water usage) for a high-quality and sustainable production.

Ceramide Phosphoethanolamine as a Possible Marker of Periodontal Disease.

Periodontal disease is a chronic oral inflammatory disorder initiated by pathobiontic bacteria found in dental plaques-complex biofilms on the tooth surface. The disease begins as an acute local inflammation of the gingival tissue (gingivitis) and can progress to periodontitis, which eventually leads to the formation of periodontal pockets and ultimately results in tooth loss. The main problem in periodontology is that the diagnosis is based on the assessment of the already obvious tissue damage. Therefore, it is necessary to improve the current diagnostics used to assess periodontal disease. Using lipidomic analyses, we show that both crucial periodontal pathogens, Porphyromonas gingivalis and Tannerella forsythia, synthesize ceramide phosphoethanolamine (CPE) species, membrane sphingolipids not typically found in vertebrates. Previously, it was shown that this particular lipid can be specifically detected by an aegerolysin protein, erylysin A (EryA). Here, we show that EryA can specifically bind to CPE species from the total lipid extract from P. gingivalis. Furthermore, using a fluorescently labelled EryA-mCherry, we were able to detect CPE species in clinical samples of dental plaque from periodontal patients. These results demonstrate the potential of specific periodontal pathogen-derived lipids as biomarkers for periodontal disease and other chronic inflammatory diseases.

Ceramide Aminoethylphosphonate as a New Molecular Target for Pore-Forming Aegerolysin-Based Protein Complexes.

Ostreolysin A6 (OlyA6) is a 15 kDa protein produced by the oyster mushroom (Pleurotus ostreatus). It belongs to the aegerolysin family of proteins and binds with high affinity to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with its partnering protein with the membrane-attack-complex/perforin domain, pleurotolysin B (PlyB), OlyA6 can form bicomponent 13-meric transmembrane pores in artificial and biological membranes containing the aegerolysin lipid receptor, CPE. This pore formation is the main underlying molecular mechanism of potent and selective insecticidal activity of OlyA6/PlyB complexes against two economically important coleopteran plant pests: the western corn rootworm and the Colorado potato beetle. In contrast to insects, the main sphingolipid in cell membranes of marine invertebrates (i.e., molluscs and cnidarians) is ceramide aminoethylphosphonate (CAEP), a CPE analogue built on a phosphono rather than the usual phosphate group in its polar head. Our targeted lipidomic analyses of the immune cells (hemocytes) of the marine bivalve, the mussel Mytilus galloprovincialis, confirmed the presence of 29.0 mol% CAEP followed by 36.4 mol% of phosphatidylcholine and 34.6 mol% of phosphatidylethanolamine. Further experiments showed the potent binding of OlyA6 to artificial lipid vesicles supplemented with mussel CAEP, and strong lysis of these vesicles by the OlyA6/PlyB mixture. In Mytilus haemocytes, short term exposure (max. 1 h) to the OlyA6/PlyB mixture induced lysosomal membrane destabilization, decreased phagocytic activity, increased Annexin V binding and oxyradical production, and decreased levels of reduced glutathione, indicating rapid damage of endo-lysosomal and plasma membranes and oxidative stress. Our data suggest CAEP as a novel high-affinity receptor for OlyA6 and a target for cytolytic OlyA6/PlyB complexes.

Insect cold-tolerance and lipidome: Membrane lipid composition of two chironomid species differently adapted to cold.

Cell-membrane fluidity is a fundamental parameter in cold resistance. It is regulated by a fine tuning of lipid composition, usually involving a great chemical diversity among head-groups, chain lengths, and degree of unsaturation. To give new insights on Alpine chironomid cold adaptation, we analysed the lipid membrane composition of Diamesa tonsa and Pseudodiamesa branickii, two species known to have different cold-tolerance, stronger in the former. Membrane lipid composition was analysed by NMR and HPLC-MS in larvae under natural (4 °C) and laboratory conditions (30 min at - 4 °C). In both species the major class of membrane lipids were phosphatidylethanolamine (PE), reaching 93% in D. tonsa and 80% in P. branickii, followed by a minor relative amount of phosphatidylcholine (PC). Phospholipids (PL) acyl chains were highly unsaturated given the presence of a relevant amount of polyunsaturated fatty acid (PUFA), among which a high proportion of ω-3 chains. This study demonstrated that these species have a similar lipidome (e.g. relevant amount of PUFA and predominance of PE), but with relevant differences on which to base different membrane fluidity: (i) a higher unsaturation index and chain length of both PE and PC and a higher ratio PE/PC ratio in D. tonsa than in P. branickii; (ii) the absence of modifications in the lipid composition in D. tonsa under sub-zero temperature. These differences might support the different cold-tolerance of the two species. In fact, we suggest that the high PE/PC ratio and the low sterols content (as in D. tonsa) could be involved in the formation of highly deformable membranes increasing their capacity to survive freezing. Interestingly, LC-MS analysis of D. tonsa lipidome revealed a new class of lipids that we named 'PpC', absent in P. branickii, that is worth investigating.

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm.

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

Early and Late Steps of Quinine Biosynthesis.

The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3. Additionally, the discovery of an O-methyltransferase specific for 6'-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.

Investigating the biosynthesis of Sch-642305 in the fungus Phomopsis sp. CMU-LMA.

Sch-642305 is an unusual bicyclic 10-membered macrolide produced by the filamentous fungus Phomopsis sp. CMU-LMA for which no biosynthetic evidence exists. Here, we generate a draft genome sequence of the producing organism and discover the biosynthetic gene cluster responsible for formation of Sch-642305. Targeted gene disruptions together with reconstitution of the pathway in the heterologous host Aspergillus oryzae dissect key chemical steps and shed light on a series of oxidoreductions occuring in the pathway.

Structural Revision and Biosynthesis of the Fungal Phytotoxins Phyllostictines A and B.

The structure of the fungal phytotoxins known as the phyllostictines has been revised to a series of bicyclic 3-methylene tetramic acids. Genome sequencing of the producing organism Phyllostica cirsii has revealed a biosynthetic gene cluster responsible for the biosynthesis of the phyllostictines, and targeted knockout experiments have proven the link and produced an intermediate.