Apart from the psychedelic psilocybin, the metabolite spectrum of Psilocybe "magic mushrooms" comprises sesquiterpenes, a class of natural products known to exhibit receptor-modulating bioactivities. However, the composition of the sesquiterpene profile has largely remained an open question. Here, we report the characterization of five Psilocybe cubensis sesquiterpene synthases, both in vitro using recombinantly produced enzymes and in vivo in Aspergillus niger. CubF is a clade I α-muurolol synthase. The investigated clade IV synthases were the near-identical CubG1 and CubG2 synthases, which catalyze mainly epi-isozizaene and β-duprezianene formation. Furthermore, CubH and CubI were identified as primarily making dauca-4(11),8-diene and β-barbatene, respectively. Gas chromatographic analyses of the headspaces of P. cubensis vegetative mycelium and fruiting bodies showed qualitative and quantitative differences, with sterpurene being among the major compounds in mycelium and dauca-4(11),8-diene in fruiting bodies. This fundamental knowledge of the P. cubensis terpenome may help distinguish the pharmacological effects of magic mushrooms versus pure psilocybin.

Psilocybe "magic mushrooms" are best known for their indolethylamine psilocybin, yet they encode enzymes for a much more diverse arsenal of small and potentially bioactive molecules. Herein, four Psilocybe cubensis clade III sesquiterpene synthases, CubB-CubE, whose genes are differently expressed in fruiting bodies compared to vegetative mycelium are reported. CubB-CubE were functionally characterized in vitro by product formation assays with heterologously produced enzymes and in vivo by transgene expression in Aspergillus niger, followed by extensive gas chromatography-mass spectrometry analyzes. CubB was identified as a single product (3R,6E)-(-)-nerolidol synthase. CubC is a multiproduct enzyme producing β-caryophyllene, β-elemene, α-humulene, and β-farnesene. CubD and CubE catalyze (near-)exclusively sterpurene formation. P. cubensis young fruiting bodies and vegetative mycelium were analyzed for sesquiterpenes, which verified the presence of the CubB product α-(3R,6E)-(-)-nerolidol. As various Psilocybe species encode highly similar enzymes, this study contributes generally to the as-yet little-understood secondary metabolome of the genus.

Ants typically demonstrate a high level of complex social behavior that is largely mediated by chemical communication. In recent years, the Clonal raider ant Ooceraea biroi has become a promising model system for the study of social behavior in ants. Here we report the profile of extracted volatiles from O. biroi and, following detection of an α-homofarnesene as the major component, unambiguously confirm its structural identity as (3Z,6E)-14-methyl-α-farnesene through preparative stereoselective total synthesis.

Little is known about the potential responses of ecologically specialized tropical species to atmospheric change and global warming. In 2 years of greenhouse experiments simulating climate change impacts, we quantified the effects of mean ambient temperature, elevated temperature (Te), current ambient CO2 concentration ([CO2]a), and doubled CO2 concentration ([CO2]e) on biomass, growth rate, and foliar chemistry of the morning glory vine, Camonea umbellata. In addition, we measured the impacts of climate change simulations on the performance and survival of the tortoise beetle, Acromis sparsa, which feeds exclusively on C. umbellata. Full-sib A. sparsa larval broods were divided into cohorts. Each cohort was placed in one of four temperature-CO2 controlled chambers and fed leaves grown in their respective treatments. Vines growing in [CO2]e more than doubled their biomass and their leaves expanded faster. The [CO2]a and Te treatments interacted to yield the greatest foliar [C]. Vines in [CO2]e and Te had the greatest C:N ratios, the lowest availability of nitrogen, and highest larval mortality. Whereas pupae were smaller and suffered lower survival in Te, pupal mass and survival increased in both the [CO2]e and Te treatments. Overall, the simultaneous elevation of both [CO2] and temperature caused declines in host quality, larval survivorship, and pupal mortality that were not observed when only one climate factor was altered. Based on this first tropical experimental study, we predict that C. umbellata will benefit from elevation of temperature and atmospheric [CO2] by altering its foliar chemistry to the detriment of its diet-specialized herbivore enemy.

Psilocybe '' magic mushrooms '' are chemically well understood for their psychotropic tryptamines. However, the diversity of their other specialized metabolites, in particular terpenoids, has largely remained an open question. Yet, knowledge on the natural product background is critical to understand if other compounds modulate the psychotropic pharmacological effects. CubA, the single clade II sesquiterpene synthase of P. cubensis, was heterologously produced in Escherichia coli and characterized in vitro, complemented by in vivo product formation assays in Aspergillus niger as a heterologous host. Extensive GC-MS analyses proved a function as multi-product synthase and, depending on the reaction conditions, cubebol, ss-copaene, delta-cadinene, and germacrene D were detected as the major products of CubA. In addition, mature P. cubensis carpophores were analysed chromatographically which led to the detection of ss-copaene and delta-cadinene. Enzymes closely related to CubA are encoded in the genomes of various Psilocybe species. Therefore, our results provide insight into the metabolic capacity of the entire genus.

Quinolinic carboxylic acids are known for their metal ion chelating properties in insects, plants and bacteria. The larval stages of the lepidopteran pest, Spodoptera littoralis, produce 8-hydroxyquinoline-2-carboxylic acid (8-HQA) in high concentrations from tryptophan in the diet. At the same time, the larval midgut is known to harbor a bacterial population. The motivation behind the work was to investigate whether 8-HQA is controlling the bacterial community in the gut by regulating the concentration of metal ions. Knocking out the gene for kynurenine 3-monooxygenase (KMO) in the insect using CRISPR/Cas9 eliminated production of 8-HQA and significantly increased bacterial numbers and diversity in the larval midgut. Adding 8-HQA to the diet of knockout larvae caused a dose-dependent reduction of bacterial numbers with minimal effects on diversity. Enterococcus mundtii dominates the community in all treatments, probably due to its highly efficient iron uptake system and production of the colicin, mundticin. Thus host factors and bacterial properties interact to determine patterns of diversity and abundance in the insect midgut.

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Its incorporation in seedlings was shown by 34S labeling experiment. Under sulfur deficiency, TMTM downregulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

The obligate pollination mutualism between Yucca and yucca moths is a classical example of coevolution. Oviposition and active pollination by female yucca moths occur at night when Yucca flowers are open and strongly scented. Thus, floral volatiles have been suggested as key sensory signals attracting yucca moths to their host plants, but no bioactive compounds have yet been identified. In this study, we showed that both sexes of the pollinator moth Tegeticula yuccasella are attracted to the floral scent of the host Yucca filamentosa. Chemical analysis of the floral headspace from six Yucca species in sections Chaenocarpa and Sarcocarpa revealed a set of novel tetranorsesquiterpenoids putatively derived from (E)-4,8-dimethyl-1,3,7-nonatriene. Their structure elucidation was accomplished by NMR analysis of the crude floral scent sample of Yucca treculeana along with GC/MS analysis and confirmed by total synthesis. Since all these volatiles are included in the floral scent of Y. filamentosa, which has been an important model species for understanding the pollination mutualism, we name these compounds filamentolide, filamentol, filamental, and filamentone. Several of these compounds elicited antennal responses in pollinating (Tegeticula) and non-pollinating (Prodoxus) moth species upon stimulation in electrophysiological recordings. In addition, synthetic (Z)-filamentolide attracted significant numbers of both sexes of two associated Prodoxus species in a field trapping experiment. Highly specialized insect-plant interactions, such as obligate pollination mutualisms, are predicted to be maintained through "private channels" dictated by specific compounds. The identification of novel bioactive tetranorsesquiterpenoids is a first step in testing such a hypothesis in the Yucca-yucca moth interaction.

Phytochemical investigation of the ethanol extract of underground parts of Iris tenuifolia Pall. afforded five new compounds; an unusual macrolide termed moniristenulide (1), 5-methoxy-6,7-methylenedioxy-4-O-2′-cycloflavan (2), 5,7,2′,3′-tetrahydroxyflavanone (3), 5-hydroxy-6,7-dimethoxyisoflavone-2′-O-β-d-glucopyranoside (9), 5,2′,3′-dihydroxy-6,7-dimethoxyisoflavone (10), along with seven known compounds (4–8, 11–12). The structures of all purified compounds were established by analysis of 1D and 2D NMR spectroscopy and HR-ESI-MS. The antimicrobial activity of the compounds 1–3, 5, 9, and 10 was investigated using the agar diffusion method against fungi, Gram-positive and Gram-negative bacteria. In consequence, new compound 3 was found to possess the highest antibacterial activity against Enterococcus faecalis VRE and Mycobacterium vaccae. Cell proliferation and cytotoxicity tests were also applied on all isolated compounds and plant crude extract in vitro with the result of potent inhibitory effect against leukemia cells. In particular, the newly discovered isoflavone 10 was active against both of the leukemia cells K-562 and THP-1 while 4–6 of the flavanone type compounds were active against only THP-1.

Plants perceive and respond to volatile signals in their environment. Herbivore-infested plants release volatile organic compounds (VOCs) which can initiate systemic defense reactions within the plant and contribute to plant-plant communication. Here, for Ipomoea batatas (sweet potato) leaves we show that among various herbivory-induced plant volatiles, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) had the highest abundance of all emitted compounds. This homoterpene was found being sufficient for a volatile-mediated systemic induction of defensive Sporamin protease inhibitor activity in neighboring sweet potato plants. The systemic induction is jasmonate independent and does not need any priming-related challenge. Induced emission and responsiveness to DMNT is restricted to a herbivory-resistant cultivar (Tainong 57), while a susceptible cultivar, Tainong 66, neither emitted amounts comparable to Tainong 57, nor showed reaction to DMNT. This is consistent with the finding that Spodoptera larvae feeding on DMNT-exposed cultivars gain significantly less weight on Tainong 57 compared to Tainong 66. Our results indicate a highly specific, single volatile-mediated plant-plant communication in sweet potato.

Induction of jasmonate-mediated plant defense against insect herbivory is initiated by a combination of both mechanical wounding and chemical factors. In order to study both effects independently on plant defense induction, SpitWorm, a computer-controlled device which mimics the damage pattern of feeding insect larvae on leaves and, in addition, can apply oral secretions (OS) or other solutions to the 'biting site' during 'feeding,' was developed and evaluated. The amount of OS left by a Spodoptera littoralis larva during feeding on Phaseolus lunatus (lima bean) leaves was estimated by combining larval foregut volume, biting rate, and quantification of a fluorescent dye injected into the larvae's foregut prior to feeding. For providing OS amounts by SpitWorm equivalent to larval feeding, dilution and delivery rate were optimized. The effectiveness of SpitWorm was tested by comparing volatile organic compounds (VOC) emissions of P. lunatus leaves treated with either SpitWorm, MecWorm, or S. littoralis larvae. Identification and quantification of emitted VOCs revealed that SpitWorm induced a volatile bouquet that is qualitatively and quantitatively similar to herbivory. Additionally, RT-qPCR of four jasmonic acid responsive genes showed that SpitWorm, in contrast to MecWorm, induces the same regulation pattern as insect feeding. Thus, SpitWorm mimics insect herbivory almost identically to real larvae feeding.

The cabbage stem flea beetle (Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the β-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala, indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

The chemical composition and antimicrobial activity of the essential oil from the aerial parts of Pyrethrum pulchrum Ledeb. were investigated. Dried plant material was hydro-distillated yielding 0.1% of essential oil. The oil was analyzed by GC-MS techniques. Fifty-five compounds were identified representing 99.7% of the total oil composition. Camphor was the predominant compound (33.9%) followed by linalool (21.1%) and α-pinene (9.0%). The antimicrobial activity of the oil was determined using the disk diffusion method against Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), Mycobacterium vaccae and fungi (Candida albicans, Sporidiobolus salmonicolor and Penicillum notatum). The essential oil of P. pulchrum displays an intermediate activity against selected bacteria.

Mohamed Abdel-Wanis Fahad Abduljabbar Matthew Abola Islam Abouyoussef Emre Acaroglu Owoicho Adogwa Komang Agung Irianto Dong Ah Shin Imad Ahmad Ashfaq Ahmed Md Kamrul Ahsan Tamir Ailon Akif Albayrak Eibar Aldana Ilyas Aleem Abduljabbar Alhammoud Mohammed Alhashash Alhashash Marjan Alimi Khalid Alsaleh Ali Andalib Andreas Andresen Prokopis Annis Petros Antonarakos A Ardeshiri Mohammed Aref Raul Arjona Paul Arnold Varun Arvind Bryan Ashman Nelson Astur John Attenello Basem Awad Ufuk Aydinli Selim Ayhan Saleh Baeesa Carlos Bagley Evan Baird Alexander Baker Philippe Bancel Giovanni Barbanti Brodano Ilaria Barni Sean Barry Paloma Bas Teresa Bas Alfonso Basurco Carlos Bautista Jiménez Pedro Bazán Drew Bednar Constanza Bedoya Benjamin Bjerke Andrey Bokov Barrett Boody Matias Borensztein Aju Bosco James Bourne Sergio Brambilla Richard Bransford Darrel Brodke Rachel Bronheim Avery Buchholz Alexander Burcev Zorica Buser Mario Cahueque Francis Camillo John Caridi Daniel Carr David Casper John Tristan Cassidy Andrew Chan Roberto Chapa Saad Chaudhary Prakash Chavan Mohammadreza Chehrassan Kazuhiro Chiba Samuel Cho Woojin Cho Woojin Cho Dean Chou Andrew Chung Andres Cobar Netzer Cordula Cristhian Cruz Bradford Currier Marcin Czyz Scott Daffner Alan Daniels Evan Davies Johannes Davis Nicolas Dea Edward DelSole Julius Dengler J. DePasse Peter Derman John DeVine Perry Dhaliwal John Di Capua Mario Diaz Donald Dietze John Dimar Herman Dittmar Ashish Diwan Tamas Doczi Pasquale Donnarumma Stephan Duetzmann John Duff Srikanth Reddy Dumpa Robert Dunn Wesley Durand Tarun Dusad Shumayou Dutta Yossry El Hawary Mohamed El-Meshtawy Aladine Elsamadicy Peter Eltes Arash Emami Richard Emery Juan Emmerich Jose Estrada Mastache Abdulfattah Ewais Asdrubal Falavigna Michael Faloon Miguel Farfan Christian Fisahn Guy Fogel Daryl Fourney John France Global Spine Journal 2018, Vol. 8(2) 209-211 a The Author(s) 2018 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/2192568218766537 journals.sagepub.com/home/gsj

Manef Abderrabba Abha Agnihotri Andrew Allan Jean-Michel Ane Emilia L. Apostolova Klaus Appenroth Birgit Arnholdt-Schmitt Ricardo Aroca Isabel Arrillaga Folkard Asch Bayram Atasagun Keiichi Baba Shashi Babbar Frantisek Baluska Balazs Barna D. Bartels Carlos Bartoli Brigitta Basa Chris Beadle Ludger Beerhues Andres Belver Oliver Berkowitz Cincia Bertea Christine Beveridge Melike Bor Marco Borghetti Rubén Bottini Fred Bourland David Burritt Jim Burton Catalina Cabot Edward Calabrese David Chagne Subbaiah Chalivendra W.C. Chang Chiew Foan Chin Jen-Ping Chung Michael Cohen M.G.C. Costa Evelyne Costes Ashton Cowan Bert Cregg Katrien Curvers Riccardo d’Andria J. Davies Peter Davies Geert-Jan De Klerk Shantaraj Deepak C.A. Delatorre Carole Deleu Lie Deng Ming-Hua Deng Renu Deswal Georg Dittie Sidzabda Djibril Dayamba Judit Dobránszki Robert Donaldson Maria Drincovich Uwe Druege Qiaohong Duan Stephen Duke Jacques Dumais Paula Duque Rivka Elbaum J. Fettke Matthew Fidelibus David Fleisher Eloise Foo Steffi Forman Vasileios Fotopoulos Christine Foyer Philipp Franken Stephen Fry Masayuki Fujita Amy Fulcher Jean-Philippe Galaud Xiang Gao Neera Garg Norberto Gariglio Christoph Geilfus Mateja Germ Jan M. Geuns Bernard Glick Eliezer Goldschmidt Albensio Gomes Silveira H. Gong J.H. Graham William Gray K. Groten Matthias Gube Guang-Qin Guo I. Guzman Edward Gwozdz Amjad Hameed H. Hartmann Yrjo Helariutta

During the process of terpene biosynthesis, C-C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the 'biosynthetic history' of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ13 C value for FDP, based on δ13 C values and relative amounts of the products, was identical with its measured δ13 C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon-carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without 'touching' the plant.

Most terrestrial carnivorous plants are specialized on insect prey digestion to obtain additional nutrients. Few species of the genus Nepenthes developed mutualistic relationships with mammals for nitrogen supplementation. Whether dietary changes require certain enzymatic composition to utilize new sources of nutrients has rarely been tested. Here, we investigated the role of urease for Nepenthes hemsleyana that gains nitrogen from the bat Kerivoula hardwickii while it roosts inside the pitchers. We hypothesized that N. hemsleyana is able to use urea from the bats' excrements. In fact, we demonstrate that 15N-enriched urea provided to Nepenthes pitchers is metabolized and its nitrogen is distributed within the plant. As ureases are necessary to degrade urea, these hydrolytic enzymes should be involved. We proved the presence and enzymatic activity of a urease for Nepenthes plant tissues. The corresponding urease cDNA from N. hemsleyana was isolated and functionally expressed. A comprehensive phylogenetic analysis for eukaryotic ureases, including Nepenthes and five other carnivorous plants' taxa, identified them as canonical ureases and reflects the plant phylogeny. Hence, this study reveals ureases as an emblematic example for an efficient, low-cost but high adaptive plasticity in plants while developing a further specialized lifestyle from carnivory to coprophagy.

Insects often rely on olfaction to communicate with conspecifics. While the chemical language of insects has been deciphered in recent decades, few studies have assessed how changes in atmospheric greenhouse gas concentrations might impact pheromonal communication in insects. Here, we hypothesize that changes in the concentration of atmospheric carbon dioxide affect the whole dynamics of alarm signaling in aphids, including: (1) the production of the active compound ( E )-β-farnesene (Eβf), (2) emission behavior when under attack, (3) perception by the olfactory apparatus, and (4) the escape response. We reared two strains of the pea aphid, Acyrthosiphon pisum , under ambient and elevated CO 2 concentrations over several generations. We found that an increase in CO 2 concentration reduced the production (i.e., individual content) and emission (released under predation events) of Eβf. While no difference in Eβf neuronal perception was observed, we found that an increase in CO 2 strongly reduced the escape behavior expressed by an aphid colony following exposure to natural doses of alarm pheromone. In conclusion, our results confirm that changes to greenhouse gases impact chemical communication in the pea aphid, and could potentially have a cascade effect on interactions with higher trophic levels.

The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer (Melolontha hippocastani), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13C cellulose and 15N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13C cellulose- and 15N urea labeled bacteria. The incorporation of 15N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

In the regurgitate (foregut content) of Spodoptera larvae we found high concentrations (0.5-5 mM) of 8-hydroxyquinoline-2-carboxylic acid (8-HQA). In a survey of different lepidopteran species, this compound was only detected in species belonging to the family of Noctuidae. 8-HQA was shown to derive from tryptophan metabolism. The amount of 8-HQA in the regurgitate was strongly dependent on the tryptophan content of the diet. In the insect 8-HQA is generated from tryptophan via kynurenine and 3-hydroxykynurenine. 8-HQA is produced by the larvae and not by their commensal gut bacteria. Analysis of different life stages of Spodoptera larvae revealed that 8-HQA is formed during the larval stage, probably acting as an iron chelator to control the gut microbiome.