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Against the context of global wildlife declines, targeted mitigation strategies have become critical to preserve what remains of biodiversity. However, the effective development of conservation too ...

International audience; Recent improvements in taxonomy consider multiple operational criteria. Integrative taxonomy provides a methodological framework merging these multisource approaches. Bumblebees are considered as a uniform group where their taxonomy remains one of the most difficult. Here, we investigate the taxonomic statuses inside a monophyletic group including six taxa (B. inexspectatus, B. mlokosievitzii, B. ruderarius, B. sylvarum, B. velox and B. veteranus)in the most diverse subgenus of bumblebees: Thoracobombus. We used an integrative approach based on mitochondrial and nuclear genetic markers and eco-chemical traits commonly used in bumblebee taxonomy. For all species, our study shows a clear differentiation in DNA and eco-chemical traits. However, we conserve the subspecies status of B. ruderarius simulatilis and B. sylvarum daghestanicus from the east of Turkey and Iran according to their degree of genetic and eco-chemical differentiation

Males of many bumblebee species exhibit a conspicuous pre-mating behavior with two distinct behavioral components: scent marking and patrol flying. The marking pheromone is produced by the cephalic part of the labial gland (CLG). As far as is known, the CLG secretion is species specific, and it usually consists of two types of compounds: (i) straight-chain aliphatic alcohols, aldehydes or esters, and (ii) acyclic mono-, sesqui- and diterpenes (alcohols or acetates). Here, we summarize data from the literature reporting chemical composition of the CLG secretions of more than 80 bumblebee species. Similarities and differences within and between subgenera are discussed in the context of biosynthetic pathways and evolution.

Background In this paper, the term “human scent signature” refers to a group of chemical compounds in a human scent trace that are in certain relative concentrations that make an unambiguous identification of persons possible. At the present time, the scent identification of persons is performed using specially trained canines. However, the scent identification mechanism as well as the molecular composition of the human scent signature is entirely unrecognized. Delimitations of the group of chemical compounds according to their volatilities that specially trained canines use for the scent identification of persons were initially the main aim of this study. Methods Eleven volunteer scent samples were split into three groups of chemical compounds, i.e., into three disjointed scent fractions with different volatilities of the compounds via preparative gas chromatography. The human scent signatures were searched in these separated scent fractions using six specially trained canines (female German Shepherds). Results The canines were able to identify individual persons successfully using different scent fractions. From this fact, it follows that one scent trace can contain more than only one group of scent compounds that can be used for scent identification by the trained canines. This observed phenomenon was named as “the multiplicity of the human scent signature” in this study. The scent identification line-up procedure provided the following results: The poorly volatile fraction resulted in an 85% success rate in the scent identification procedure. The middle fraction resulted in a 58% success rate, and the highly volatile fraction resulted in a 30% success rate. Conclusions In this study, it was found that the human scent sample contains more than one group of scent compounds; according to which the trained canines are able to identify human individuals. It means that the human scent sample contains more than only one scent signature. The authors of this research believe that results of this study will open a broad discussion about the multiplicity of the human scent signature, primary scent molecules, optimal canine training, and in general, the scent identification method in many countries.

International audience; Multisource approaches in taxonomy gather different lines of evidence in order to draw strongly supported taxonomic conclusions and constitute the basis of integrative taxonomy. In the case of overlooked taxa with disjunct distributions for which sampling is more challenging, integrative approaches help to propose stable hypotheses at the species and subspecies levels. Here, based on genetic and semio-chemical traits, we performed an integrative taxonomic analysis to evaluate species delimitation hypotheses within a monophyletic group of bumblebees (Hymenoptera, Apidae, Bombus) including the formerly recognised subgenera Eversmannibombus, Laesobombus and Mucidobombus which are now included in the subgenus Thoracobombus. Our results demonstrate the conspecificity of several polytypic taxa, and we formally recognise the subspecies Bombus laesus aliceae comb. nov. Cockerell, 1931, endemic to North Africa, based on its allopatry, unique mitochondrial haplotype and divergent cephalic labial gland secretions. This highlights the need to maintain studying polytypic complexes of bumblebee taxa for which phylogenetic relationships could be still entangled and eventually implement conservation strategies for taxonomically differentiated lineages.

The bumblebee wax moth, Aphomia sociella, is a parasite of bumblebees. In this species, males produce sexual pheromone to attract females, while females produce an aphrodisiac pheromone that initiates male courtship. Both pheromones contain 6,10,14-trimethylpentadecan-2-one (TMPD-one) and the corresponding alcohol, 6,10,14-trimethylpentadecan-2-ol (TMPD-ol) in sex specific quantities. Male sex pheromone consists of 7 components with TMPD-one as a minor one and traces of TMPD-ol. In female aphrodisiac pheromone, TMPD-ol is the major component, while TMPD-one is present in traces. Here we report on the absolute configuration of TMPD-one in male sex pheromone and TMPD-ol in female aphrodisiac pheromone of A. sociella. The configuration was determined from GC/MS of prepared (S)-acetoxypropionyl esters of TMPD-ol. TMPD-one was first reduced to the alcohol that was then derivatized with (S)-acetoxypropionyl chloride. The GC/MS data of obtained diastereoisomers were compared with synthetic standards. The absolute configuration of TMPD-one in the male pheromone was (6R,10R). The configuration of TMPD-ol in the female pheromone was (2R,6R,10R). Electrophysiological experiments showed that TMPD-one and TMPD-ol are perceived by both sexes. The synthetic standards of naturally produced stereoisomers elicited higher responses than mixtures of all stereoisomers. We are grateful to J. Titzenthalerová for her skillful assistance in electrophysiological experiments and to R. Hanus for his help with statistical evaluations of the EAG recordings. This work was financially supported by the Ministry of Education of the Czech Republic (grant No. LD15102). The research was done within the frames of the COST Action Super-B (FA1307). EW and EH are thankful for the funding from ERUF (European regional developmental fund), the region of Jämtland/Härjedalen and the region of Västernorrland.

Cold-adapted species are expected to have reached their largest distribution range during a part of the Ice Ages whereas postglacial warming has led to their range contracting toward high-latitude ...

There are more than one million described insect species. This species richness is reflected in the diversity of insect metabolic processes. In particular, biosynthesis of secondary metabolites, such as defensive compounds and chemical signals, encompasses an extraordinarily wide range of chemicals that are generally unparalleled among natural products from other organisms. Insect genomes, transcriptomes and proteomes thus offer a valuable resource for discovery of novel enzymes with potential for biotechnological applications. Here, we focus on fatty acid (FA) metabolism-related enzymes, notably the fatty acyl desaturases and fatty acyl reductases involved in the biosynthesis of FA-derived pheromones. Research on insect pheromone-biosynthetic enzymes, which exhibit diverse enzymatic properties, has the potential to broaden the understanding of enzyme specificity determinants and contribute to engineering of enzymes with desired properties for biotechnological production of FA derivatives. Additionally, the application of such pheromone-biosynthetic enzymes represents an environmentally friendly and economic alternative to the chemical synthesis of pheromones that are used in insect pest management strategies.

A fast, inexpensive, and ecological method based on liquid-liquid extraction principle has been developed for preparation of extract with high content of Nigella sativa (N. sativa) quinones. The gas chromatography-mass spectrometry (GC-MS) analysis showed that oil extraction using 70% ethanol for 6 h yielded extract containing 50% of thymoquinone (TQ) and 13% of thymohydroquinone (THQ).

Bumblebees have been the focus of much research, but the taxonomy of many species groups is still unclear, especially for circumpolar species. Delimiting species based on multisource datasets provides a solution to overcome current systematic issues of closely related populations. Here, we use an integrative taxonomic approach based on new genetic and eco-chemical datasets to resolve the taxonomic status of Bombus lapponicus and Bombus sylvicola. Our results support the conspecific status of B. lapponicus and B. sylvicola and that the low gradual divergence around the Arctic Circle between Fennoscandia and Alaska does not imply speciation in this species complex. Therefore, based on our molecular and morphological analyses, we propose to assign them subspecific status: Bombus lapponicus lapponicus from Fennoscandia and West Siberia and Bombus lapponicus sylvicola comb. nov. from Alaska and Yukon. In addition, our analyses reveal a cryptic species in the B. lapponicus complex from Alaska, which we describe here as new: Bombus (Pyrobombus) interacti sp. nov.

Social parasitic Hymenopterans have evolved morphological, chemical, and behavioral adaptations to overcome the sophisticated recognition and defense systems of their social host to invade host nests and exploit their worker force. In bumblebees, social parasitism appeared in at least 3 subgenera independently: in the subgenus Psithyrus consisting entirely of parasitic species, in the subgenus Alpinobombus with Bombus hyperboreus, and in the subgenus Thoracobombus with B. inexspectatus. Cuckoo bumblebee males utilize species-specific cephalic labial gland secretions for mating purposes that can impact their inquiline strategy. We performed cephalic labial gland secretions in B. hyperboreus, B. inexspectatus and their hosts. Males of both parasitic species exhibited high species specific levels of cephalic gland secretions, including different main compounds. Our results showed no chemical mimicry in the cephalic gland secretions between inquilines and their host and we did not identify the repellent compounds already known in other cuckoo bumblebees.

The current bumblebee decline leads to inbreeding in populations that fosters a loss of allelic diversity and diploid male production. As diploid males are viable and their offspring are sterile, bumblebee populations can quickly fall in a vortex of extinction. In this article, we investigate for the first time a potential premating mechanism through a major chemical reproductive trait (male cephalic labial gland secretions) that could prevent monandrous virgin queens from mating with diploid males. We focus our study on the cephalic labial gland secretions of diploid and haploid males of Bombus terrestris (L.). Contrary to initial expectations, our results do not show any significant differentiation of cephalic labial gland secretions between diploid and haploid specimens. Queens seem therefore to be unable to avoid mating with diploid males based on their compositions of cephalic labial gland secretions. This suggests that the vortex of extinction of diploid males could not be stopped through premating avoidance based on the cephalic labial gland secretions but other mechanisms could avoid mating between diploid males and queens.

Male marking pheromones (MPs) are used by the majority of bumblebee species (Hymenoptera: Apidae), including a commercially important greenhouse pollinator, the buff-tailed bumblebee (Bombus terrestris), to attract conspecific females. MP biosynthetic processes in the cephalic part of the bumblebee male labial gland (LG) are of extraordinary complexity, involving enzymes of fatty acid and isoprenoid biosynthesis, which jointly produce more than 50 compounds. We employed a differential transcriptomic approach to identify candidate genes involved in MP biosynthesis by sequencing Bombus terrestris LG and fat body (FB) transcriptomes. We identified 12 454 abundantly expressed gene products (reads per kilobase of exon model per million mapped reads value > 1) that had significant hits in the GenBank nonredundant database. Of these, 876 were upregulated in the LG (> 4-fold difference). We identified more than 140 candidate genes potentially involved in MP biosynthesis, including esterases, fatty acid reductases, lipases, enzymes involved in limited fatty acid chain shortening, neuropeptide receptors and enzymes involved in biosynthesis of triacylglycerols, isoprenoids and fatty acids. For selected candidates, we confirmed their abundant expression in LG using quantitative real-time reverse transcription-PCR (qRT-PCR). Our study shows that the Bombus terrestris LG transcriptome reflects both fatty acid and isoprenoid MP biosynthetic processes and identifies rational gene targets for future studies to disentangle the molecular basis of MP biosynthesis. Additionally, LG and FB transcriptomes enrich the available transcriptomic resources for Bombus terrestris.

International audience; AbstractSpecies taxonomy of bumblebees (Bombus Latreille, 1802) is well known to be problematic due to a potentially high intra-specific variability of morphological traits while different species can converge locally to the same color pattern (cryptic species). Assessing species delimitation remains challenging because it requires to arbitrarily select variable traits whose accuracy continues to be debated. Integrative taxonomic approach seems to be very useful for this group as different independent traits are assessed to propose a rational taxonomic hypothesis. Among operational criteria to assess specific status, the reproductive traits involved in the pre-mating recognition (i.e., the male cephalic labial gland secretions, CLGS) have been premium information. Since these secretions are supposed to be species-specific, these chemical traits can bring essential information where species delimitation is debated. Here, we describe and compare the CLGS of 161 male specimens of nine Alpinobombus taxa: alpinus, balteatus, helleri, hyperboreus, kirbiellus, natvigi, neoboreus, polaris, and pyrrhopygus. We aim also to test the congruence between this new information (reproductive traits) and published genetic dataset. Our results emphasized six distinct groups with diagnostic major compounds: (a) alpinus + helleri with hexadec-9-en-1-ol; (b) polaris + pyrrhopygus with two major compounds hexadec-9-en-1-ol and hexadec-9-enal; (c) balteatus with tetradecyl acetate; (d) kirbiellus with geranyl geranyl acetate; (e) hyperboreus + natvigi with octadec-11-en-1-ol; (f) neoboreus with octadec-9-en-1-ol. Based on this new information, we can confirm the species status of B. alpinus, B. balteatus, B. hyperboreus, B. kirbiellus, B. neoboreus, and B. polaris. We also confirm the synonymy of helleri (Alps) with alpinus (Sweden). However, the specific status of natvigi (Alaska) and pyrrhopygus (Sweden) is questionable and these taxa do not have specific CLGS composition.

Fatty acyl reductases (FARs) are involved in the biosynthesis of fatty alcohols that serve a range of biological roles. Insects typically harbor numerous FAR gene family members. While some FARs are involved in pheromone biosynthesis, the biological significance of the large number of FARs in insect genomes remains unclear. Using bumble bee (Bombini) FAR expression analysis and functional characterization, hymenopteran FAR gene tree reconstruction, and inspection of transposable elements (TEs) in the genomic environment of FARs, we uncovered a massive expansion of the FAR gene family in Hymenoptera, presumably facilitated by TEs. The expansion occurred in the common ancestor of bumble bees and stingless bees (Meliponini). We found that bumble bee FARs from the expanded FAR-A ortholog group contribute to the species-specific pheromone composition. Our results indicate that expansion and functional diversification of the FAR gene family played a key role in the evolution of pheromone communication in Hymenoptera., eLife digest Many insects release chemical signals, known as sex pheromones, to attract mates over long distances. The pheromones of male bumble bees, for example, contain chemicals called fatty alcohols. Each species of bumble bee releases a different blend of these chemicals, and even species that are closely related may produce very different ‘cocktails’ of pheromones. The enzymes that make fatty alcohols are called fatty acyl reductases (or FARs for short). Any change to a gene that encodes one of these enzymes could change the final mix of pheromones produced. This in turn could have far-reaching effects for the insect, and in particular its mating success. Over time, these changes could even result in new species. Yet no one has previously looked into how the genes for FAR enzymes have evolved in bumble bees, or how these genes might have shaped the evolution of this important group of insects. Tupec, Buček et al. set out to learn what genetic changes led the males of three common species of bumble bees to make dramatically different mixes of pheromones. Comparing the genetic information of bumble bees with that of other insects showed that the bumble bees and their close relatives, stingless bees, often had extra copies of genes for certain FAR enzymes. Inserting some of these genes into yeast cells caused the yeast to make the correct blend of bumble bee pheromones, confirming that these genes did indeed produce the mixture of chemicals in these signals. Further, detailed analysis of the bumble bees’ genetic information revealed many genetic sequences, called transposable elements, close to the genes for the FAR enzymes. Transposable elements make the genetic material less stable; they can be ‘cut’ or ‘copied and pasted’ in multiple locations and often cause other genes to be duplicated or lost. Tupec et al. concluded that these transposable elements led to a dramatic increase in the number of genes for FAR enzymes in a common ancestor of bumble bees and stingless bees, ultimately allowing a new pheromone ‘language’ to evolve in these insects. These results add to our understanding of the chemical and genetic events that influence what chemicals insects use to communicate with each other. Tupec, Buček et al. also hope that a better knowledge of the enzymes that insects use to make pheromones could have wide applications. Other insects – including pest moths – use a similar mixture of fatty alcohols as pheromones. Artificially produced enzymes, such as FAR enzymes, could thus be used to mass-produce pheromones that may control insect pests.

Males of the closely related species Bombus terrestris and Bombus lucorum attract conspecific females by completely different marking pheromones. MP of B. terrestris and B. lucorum pheromones contain mainly isoprenoid (ISP) compounds and fatty acid derivatives, respectively. Here, we studied the regulation of ISP biosynthesis in both bumblebees. RNA-seq and qRT-PCR analyses indicated that acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and farnesyl diphosphate synthase (FPPS) transcripts are abundant in the B. terrestris labial gland. Maximal abundance of these transcripts correlated well with AACT enzymatic activity detected in the LG extracts. In contrast, transcript abundances of AACT, HMGR, and FPPS in B. lucorum were low, and AACT activity was not detected in LGs. These results suggest that transcriptional regulation plays a key role in the control of ISP biosynthetic gene expression and ISP pheromone biosynthesis in bumblebee males.

The recent development of the integrative taxonomic approach in bumblebees has led to unexpected merging or splitting of several taxa. Here we investigate the taxonomic statuses of one of the most abundant, widespread and polytypic Palearctic bumblebees, Bombus pascuorum. The latest review of this species includes 24 subspecies. We used an integrative approach based on genetic markers and male chemical reproductive traits and compared our results with the former classifications. Our results show that all B. pascuorum taxa are conspecific and share the same male chemical reproductive traits. The genetic structure observed in one mitochondrial and two nuclear markers poorly reflects the current subspecific classification. Indeed, the concordance of population genetic differentiation, population geographic distribution, and population colour pattern similarity suggests a different meaningful prospective classification with four taxon complexes: (i) the B. pascuorum dusmeti group, including all taxa from the Iberian Peninsula and south-west France; (ii) B. pascuorum rehbinderi; (iii) B. pascuorum siciliensis; and (iv) B. pascuorum floralis groups (including all other taxa studied here).

The aim of this study was to compare the effect of different heat treatments (pan-frying, oven-baking, and grilling) on the contents of polyunsaturated fatty acids (PUFAs) in fish tissue. Four fish species were examined: pike, carp, cod, and herring. High performance liquid chromatography, coupled with electrospray ionization and mass spectrometric detection (HPLC/ESI/MS), was employed for determination of intact lipid molecules containing n-3 and n-6 PUFAs. Although mostly non-polar lipids (triacylglycerols, TGs) were present in the fish tissue, the PUFAs were present preferentially in the phospholipid fraction. Omnivorous fish species (carp, herring) contained more TGs than did predatory ones (pike, cod). Higher amounts of PUFAs were detected in the marine species than in the freshwater ones. The impact of heat treatments on the lipid composition in the fish tissue seems to be species-specific, as indicated by multivariate data analysis. Herring tissue is most heat-stable, and the mildest heat treatment for PUFA preservation was oven-baking.

Lepidoptera evolved tympanic ears in response to echolocating bats. Comparative studies have shown that moth ears evolved many times independently from chordotonal organs. With only 1 to 4 receptor cells, they are one of the simplest hearing organs. The small number of receptors does not imply simplicity, neither in behavior nor in the neural circuit. Behaviorally, the response to ultrasound is far from being a simple reflex. Moths’ escape behavior is modulated by a variety of cues, especially pheromones, which can alter the auditory response. Neurally the receptor cell(s) diverges onto many interneurons, enabling parallel processing and feature extraction. Ascending interneurons and sound-sensitive brain neurons innervate a neuropil in the ventrolateral protocerebrum. Further, recent electrophysiological data provides the first glimpses into how the acoustic response is modulated as well as how ultrasound influences the other senses. So far, the auditory pathway has been studied in noctuids. The findings agree well with common computational principles found in other insects. However, moth ears also show unique mechanical and neural adaptation. Here, we first describe the variety of moths’ auditory behavior, especially the co-option of ultrasonic signals for intraspecific communication. Second, we describe the current knowledge of the neural pathway gained from noctuid moths. Finally, we argue that Galleriinae which show negative and positive phonotaxis, are an interesting model species for future electrophysiological studies of the auditory pathway and multimodal sensory integration, and so are ideally suited for the study of the evolution of behavioral mechanisms given a few receptors.

Buff-tailed bumblebees, Bombus terrestris, use a male sex pheromone for premating communication. Its main component is a sesquiterpene, 2,3-dihydrofarnesol. This paper reports the isolation of a thiolase (acetyl-CoA thiolase, AACT_BT), the first enzyme involved in the biosynthetic pathway leading to formation of isoprenoids in the B. terrestris male sex pheromone. Characterisation of AACT_BT might contribute to a better understanding of pheromonogenesis in the labial gland of B. terrestris males. The protein was purified to apparent homogeneity by column chromatography with subsequent stepwise treatment. AACT_BT showed optimum acetyltransferase activity at pH 7.1 and was strongly inhibited by iodoacetamide. The enzyme migrated as a band with an apparent mass of 42.9 kDa on SDS-PAGE. MS analysis of an AACT_BT tryptic digest revealed high homology to representatives of the thiolase family. AACT_BT has 96 % amino acid sequence identity with the previously reported Bombus impatiens thiolase.

Two-dimensional comprehensive gas chromatography (GC×GC) coupled with mass detection was used as a tool for biosynthetic studies of bumblebee pheromones. Prior to biosynthetic experiments, the chromatographic behaviour of isotopically modified esters in the GC×GC system as well as their behaviour in mass detection was studied. The male marking pheromones of Bombus lucorum, Bombus lapidarius and Bombus terrestris were investigated. Main pheromonal components are ethyl tetradec-9-enoate (53 %) and ethyl dodecanoate (6 %) in B. lucorum, hexadec-9-en-1-ol (52 %) and hexadecan-1-ol (31 %) in B. lapidarius, and 2,3-dihydrofarnesol (58 %) and ethyl dodecanoate (15 %) in B. terrestris. The research strategy was based on 1) in vivo incubation of isotopically (2H, 13C) modified fatty acids (FAs) and analysis of their metabolites and 2) feeding experiments with 2H- and 13C-labelled FAs mixed with food. It was observed that labelled FAs were modified into the most abundant aliphatic compounds present in labial gland secretions. In feeding experiments, the labelled FAs were transformed into pheromone components. Transport of the FA precursors from the fat body through haemolymph was confirmed. The results show that FAs, stored in the form of triacylglycerols in the fat body, are likely to participate in the biosynthesis of some aliphatic pheromone components.

Sex-specific chemical secretions have been widely used as diagnostic characters in chemotaxonomy. The taxonomically confused group of bumblebees has reaped the benefit of this approach through the analyses of cephalic labial gland secretions (CLGS). Most of currently available CLGS descriptions concern species from the West-Palearctic region but few from the New World. Here, the CLGS of four East-Palearctic species Bombus deuteronymus, B. filchnerae, B. humilis, and B. exil (subgenus Thoracobombus) are analysed. Our results show high levels of variability in the major compounds in B. exil. In contrast, we describe a low differentiation in CLGS compounds between B. filchnerae and its phylogenetically closely related taxon B. muscorum. Moreover, the chemical profiles of B. filchnerae and B. muscorum are characterized by low concentrations of the C16 component, which is found in higher concentrations in the other Thoracobombus species. This raises the possibility that courtship behavior as well as environmental constraints could affect the role of the bumblebee males' CLGS.

Three bacterial strains belonging to the genus Lactobacillus were isolated from the digestive tracts of laboratory-reared bumblebee queens (Bombus terrestris) using MRS agar under anaerobic conditions. The isolates were identified according to 16S rRNA gene sequence analysis as undescribed members of the genus Lactobacillus , with the highest 16S rRNA gene sequence similarity (96.9 %) to the uncharacterized bacterial strain Lactobacillus sp. Mboho2r2 isolated from the stomach of a European honeybee (Apis mellifera). Lactobacillus tucceti was found to be the closest related species with a validly published name, with 92.9 % 16S rRNA gene sequence similarity to the type strain. However, phylogenetic analyses based on different markers revealed that this species is phylogenetically very distant from the novel strains. The DNA G+C content of the proposed type strain BTLCH M1/2T is 37.8 mol%. The fatty acids C19 : 1ω6c and/or C19 : 0 cyclo ω10c/19ω6, C18 : 1ω9c and C16 : 0 were predominant in all strains. Diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids and two phosphoglycolipids were detected in the novel strains. Growth was observed at 47 °C. The peptidoglycan type A4α l-Lys–d-Asp was determined for strain BTLCH M1/2T. Genotypic characteristics and phylogenetic analyses based on the phylogenetic markers hsp60, pheS, rpoA and tuf as well as phenotypic characteristics and the results of chemotaxonomic analyses confirmed that the new isolates belong to a novel species of the genus Lactobacillus , for which the name Lactobacillus bombi sp. nov. is proposed. The type strain is BTLCH M1/2T ( = DSM 26517T = CCM 8440T).

The enantioselective allylation of an aromatic aldehyde to give a chiral homoallylic alcohol was employed as the key step in the syntheses of a flobufen metabolite and dapoxetine. In the former case, the homoallylic alcohol moiety (99 % ee) was converted into a five-membered lactone ring with good preservation of the optical purity, and the target compound, a flobufen metabolite, was obtained in 95 % ee. In the latter case, the homoallylic alcohol moiety (97 % ee) was transformed over several steps into a 3-aminopropanol moiety. During the course of the synthesis, the gradual loss of optical purity was observed, and the target compound, dapoxetine, was obtained in 85 % ee.

Triacylglycerols (TGs) stored in the fat bodies of bumblebee males have a species-specific composition. The striking structural similarities between TG fatty acids (FAs) and components of the male marking pheromone in certain species led to the hypothesis that FAs may serve as precursors in pheromone biosynthesis. Here, we analysed TGs from B. ruderatus, B. bohemicus, and B. campestris. Nonadec-9-ene and icos-15-en-1-ol are the main components of B. ruderatus labial gland secretion, forming up to 92% of the gland extract. The corresponding icos-11-enic and icos-15-enic acids were found in TGs at levels higher than usual for bumblebee species. We found similar relationships in B. campestris and B. bohemicus. These results suggest that FAs might be precursors of aliphatic compounds in the male pheromones. Furthermore, we report for the first time the pheromone structure of B. ruderatus males.

The enantioselective allylation of thio- phene-2-carbaldehyde with allyltrichlorosilane under Lewis base catalysis has been studied. The use of cat- alytic amount (1 mol%) of chiral bipyridine N,N'-di- oxides provided the corresponding 1-(thiophen-2-yl)- but-3-en-1-ol with asymmetric induction reaching 97% ee. The prepared chiral (S)-1-(thiophen-2-yl)- but-3-en-1-ol was used as the crucial chiral building block in a formal total synthesis of duloxetine.

A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35°C using N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanine 4-nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55±0.042 mM and 0.714±0.056 μmol p-nitroalanine produced min(-1) mg protein(-1) , respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone, which indicated the trypsin-like but not chymotrypsin-like specificity. The identity of the serine protease was confirmed by nanoliquid-tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence.

Understanding the social evolution leading to insect eusociality requires, among other, a detailed insight into endocrine regulatory mechanisms that have been co-opted from solitary ancestors to play new roles in the complex life histories of eusocial species. Bumblebees represent well-suited models of a relatively primitive social organization standing on the mid-way to highly advanced eusociality and their queens undergo both, a solitary and a social phase, separated by winter diapause. In the present paper, we characterize the gene expression levels of major endocrine regulatory pathways across tissues, sexes, and life-stages of the buff-tailed bumblebee, Bombus terrestris, with special emphasis on critical stages of the queen’s transition from solitary to social life. We focused on fundamental genes of three pathways: (1) Forkhead box protein O and insulin/insulin-like signaling, (2) Juvenile hormone signaling, and (3) Adipokinetic hormone signaling. Virgin queens were distinguished by higher expression of forkhead box protein O and downregulated insulin-like peptides and juvenile hormone (JH) signaling, indicated by low expression of methyl farnesoate epoxidase (MFE) and transcription factor Kruppel homolog 1 (Kr-h1). Diapausing queens showed the expected downregulation of JH signaling in terms of low MFE and vitellogenin (Vg) expressions, but an unexpectedly high expression of Kr-h1. By contrast, reproducing queens revealed an upregulation of MFE and Vg together with insulin signaling. Surprisingly, the insulin growth factor 1 (IGF-1) turned out to be a queen-specific hormone. Workers exhibited an expression pattern of MFE and Vg similar to that of reproducing queens. Males were characterized by high Kr-h1 expression and low Vg level. The tissue comparison unveiled an unexpected resemblance between the fat body and hypopharyngeal glands across all investigated genes, sexes, and life stages.

Instrumental human scent analysis is undoubtedly desirable for many forensic as well medical applications. Most of the previous human scent studies were focused on volatile organic compounds (VOCs) which were analysed by head space solid phase micro-extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS). This method is, however, significantly less sensitive to "heavier" less volatile compounds emitted from the human skin. These less volatile organic scent molecules probably create the basis of the individual human scent signature, and therefore, our attention is focused mainly on these "heavier" compounds. The human scent was adsorbed onto purified glass beads and samples were prepared as hexane solutions obtained by extraction from the sampled glass beads. To resolve a lot of very similar molecules, the comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometer (GCxGC-TOFMS) was used to analyse the hexane scent solutions. Using this technique, more than 137 less volatile molecules including organic fatty acids, ketones, aldehydes, simple esters, alcohols, and especially various fatty acid esters with different carbon chains were identified. A considerable number of these molecules were identified in the scent samples for the first time.

International audience; The species international trade leads to multiple non-native invasions. Besides species invasions, commercial exchanges may also contribute to translocation between closely related taxa or allopatric populations. Consequently, preserving endemic taxa and specificity of local populations require to regulate commercial translocations of species or populations. To be efficient such regulation needs a resolved taxonomy and a thorough analysis of the population structure of native taxa/populations. To provide guidelines for an efficient regulation of the trade of Bombus terrestris within its natural range, we analyzed its taxonomy and its population structure using an integrative taxonomic approach. Our results show that B. terrestris translocations involve two species, three subspecies, and several populations with weak differentiation. These different levels of differentiation imply specific and appropriate regulations of translocations with different levels of prioritization. We ultimately assess the relevance of current policies and propose potentially efficient regulations for policy-makers. Such integrative taxonomic approach should be used in other traded polytypic species.

Thirteen samples of an efflorescence collected from the surface of beeswax seals or from beeswax used for their restoration were analysed in detail. The samples were of different origin, age and storage history. The composition and the incidence of the efflorescence was correlated with a chemical composition of historical and recent beeswax samples. The composition of the crystalline layer was found to be very similar in all the samples. Linear monounsaturated alkenes containing 31 and 33 carbon atoms, more precisely ( Z )-hentriacont-10-ene and ( Z )-tritriacont-10-ene, were identified as main components. The analyses of beeswax have revealed that all compounds identified in “wax bloom” naturally occur in recent beeswax. However, the historical beeswaxes contained only traces of unsaturated hydrocarbons, if anything. The efflorescence was observed primarily on the surfaces of those samples which contained larger proportion of alkenes. The presented results could be of particular importance not only for better understanding of the described phenomenon, but also for the development of new beeswax-based materials, which would be “bloom resistant” and useful for conservation purposes.

Among promising alternative methods to control food spoilage much attention is being paid to the use of essential oils (EOs), and lately also to their activity in vapor phase. Until now, no standard assay exists, and there are many methods used by different authors, but any of them is suitably adapted for fast screening of large quantities of samples. We have modified most commonly used disc volatilization method by using four-section Petri dish (PD, 90 mm diameter), large filter paper disc (85 mm diameter) evenly impregnated with EO and medium containing lid. In comparison to normal disc volatilization method, labor and material needed are several times reduced, while the composition of headspace is more uniform than in case of 6 mm disc, where different volatility of particular compounds could influence the results. Sixty nine EOs were tested for its antimicrobial activity against three bacteria (Staphylococcus aureus, Salmonella enteritidis, Pseudomonas aeruginosa) and three fungi (Alternaria alternata, Aspergillus niger, Penicillium digitatum). Thirty nine EOs inhibited at least one microorganism, Armoracia rusticana and Cinnamomum zeylanicum being the most active, followed by EOs of Eugenia caryophyllata, Origanum compactum, Origanum vulgare, Pimenta racemosa, Thymus serpyllum and Thymus vulgaris inhibiting all six or five microorganisms, respectively. According to our opinion, this method is suitable for screening of large quantities of samples for antimicrobial activity in vapor phase.

The interpretation of the electron ionization mass spectra of straight-chain and methyl-branched saturated and unsaturated wax esters (WEs) is discussed in this study based on the spectra of 154 standards. The most important fragments indicative of the structure of the acid and alcohol chains are identified and summarized for WEs with various number of double bonds in the chains. Briefly, most WEs provide acylium ions allowing structural characterization of the acid part, whereas the alcohol part gives corresponding alkyl radical cations. The elemental composition of selected important fragments is established from a high-resolution accurate mass analysis. The ion abundances are discussed with respect to the length and unsaturation of the aliphatic chains. The interpretation of the spectra of branched or unsaturated WEs requires the recognition of small but important peaks that are difficult to discern among the other fragments. We demonstrate that such fragments are easily detected in differential mass spectra. This approach requires spectra of WE standards (e.g., straight-chain analogs in the case of branched WEs) recorded under the same experimental conditions. The WEs mass spectral database provided in the supplemental data can be used as a reference for the analysis of the GC/EI-MS data.

Biotransformation of (1S)-2-carene and (1S)-3-carene by Picea abies suspension culture led to the formation of oxygenated products. (1S)-2-Carene was transformed slowly and the final product was identified as (1S)-2-caren-4-one. On the other hand, the transformation of (1S)-3-carene was rapid and finally led to the formation of (1S)-3-caren-5-one and (1S)-2-caren-4-one as equally abundant major products. The time-course of the reaction indicates that some products abundant at the beginning of the reaction (e.g. (1S, 3S, 4R)-3,4-epoxycarane and (1R)-p-mentha-1(7),2-dien-8-ol) were consumed by a subsequent transformations. Thus, a precise selection of the biotransformation time may be used for a production of specific compounds.

Three kinds of turpentine (a waste by-product of industrial wood processing) of various monoterpene compositions were transformed by Picea abies cells and the product yields monitored in relation to the initial turpentine concentration. The major products obtained were trans-verbenol, trans-pinocarveol, myrtenol, α -terpineol, and p-cymen-8-ol depending on the substrate composition. The absolute quantitative values of the major products were evaluated for a substrate concentration of 0.86 g L 1 . The concentration of trans-verbenol and trans-pinocarveol after twelve days of biotransformation was 768 and 388 mg L 1 , respectively. The substrate was uptaken by Picea abies cells within the fi rst three days and the majority of products released in fi ve days. Although not all the starting material was consumed, the Picea abies suspension culture was able to convert concentrations of turpentine as high as 4.3 g L 1 into valuable products. By precise selection of the substrate concentration and time course, favourable conversion to products could be achieved.

The aim of this study was to identify antimicrobial properties of selected essential oils in the vapour phase against different strains of Staphylococcus aureus, including MRSA and resistant clinical isolates. The resistance pattern of used S. aureus strains was checked by disc diffusion method against three antibiotics. Essential oils tested were obtained by hydro-distillation and characterized by GC-MS and SPME-GC. Their antimicrobial properties in the vapour phase were determined by the micro-atmosphere method. Among the essential oils tested, the most active was Armoracia rusticana with MICs ranging from 8.3 to 17 µl/l, followed by Origanum syriacum (8.3–130 µl/l), Allium sativum (8.3–530 µl/l), Satureja hortensis (17–130 µl/l), Satureja montana (33–260 µl/l), Thymus vulgaris (33–260 µl/l), and Thymus serpyllum (33–530 µl/l). To the best of our knowledge, this is the first report of their antimicrobial properties in vapour phase against a collection of strains and clinical isolates of S. aureus, including MRSA. Based on the results, the essential oils tested can be considered as effective anti-staphylococcal natural products with specific application possibilities due to the activity in the vapour phase. Copyright © 2011 John Wiley & Sons, Ltd.

Human cerumen was separated by column chromatography into the following groups of compounds: hydrocarbons, squalene, wax esters and cholesterol esters, triacylglycerols, free fatty acids, free fatty alcohols, monoacylglycerols, free cholesterol, free sterols, and free hydroxy acids. The groups of compounds obtained were examined in detail by gas chromatography and gas chromatography-mass spectrometry. In total, about one thousand compounds have been identified.

Amaryllidaceae are known as ornamental plants, furthermore some species of this family contain galanthamine, an acetylcholinesterase inhibitor approved for the treatment of Alzheimer's disease, and other alkaloids with interesting pharmacological activity. The chemical composition of alkaloids from Zephyranthes grandiflora Lindl. was analyzed by GC/MS. Seven known compounds, belonging to five structural types of Amaryllidaceae alkaloids, were identified. The alkaloid extract from the bulbs showed promising cholinesterase inhibitory activities against human blood acetylcholinesterase (HuAChE; IC50 39.2±3.0 µg/mL) and human plasma butyrylcholinesterase (HuBuChE; IC50 356±9.3 µg/mL).

Males of Bombus terrestris (L.) adopt a patrolling behaviour during their nuptial parade using cephalic labial gland (CLG) secretions containing sexual pheromones to attract conspecific virgin queens. The changes in chemical composition of their CLG secretions with age are quite well known. In this study, we investigate the evolution of CLG secretions with age in greater detail and compare behavioural reactions of conspecific virgin queens to the secretions. We show that compounds of CLG secretions follow two profiles. Most of the compounds increase from the first day after emergence until the bees are 15-days-old and then decrease. Others are less abundant in 1 to 15-day-old males and then increase (e.g. tricosane, tricosene, henicosane, tetradecanoic acid, pentacosene, pentacosane, heptacosene, heptacosane, nonacosene and geranylcitronellyl tetradecanoate). Differences in secretion composition lead to preferences of virgin queens for males according to the male’s age. Virgin queens prefer the pheromonal gland secretions of bees of the following ages in decreasing order; 1 day = 3 days < 7 days = 30 days < 15 days < 10 days. The virgin queens are strongly attracted by secretions containing high amounts of 2,3-dihydrofarnesol, 2,3-dihydrofarnesal, ethyl dodecanoate and hexadecanol. On the contrary, geranylcitronellol is more abundant in 30-day-old males.

Within the complex network of chemical signals used by termites, trail pheromones and sex pheromones are among the best known. Numerous recent papers map the chemical identity and glandular origin of these pheromones in nearly all major isopteran taxa. In this study, we aimed to describe the sex pheromone and the trail pheromone of a poorly known sand termite, Psammotermes hybostoma. We identified (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (dodecatrienol) as the sex pheromone released by tergal and sternal glands of female imagos and, at the same time, as the trail pheromone secreted from the sternal gland of workers. We conclude that chemical communication in Psammotermes does not differ from that of most other Rhinotermitidae, such as Reticulitermes, despite the presence of a diterpene as a major component of the trail pheromone of Prorhinotermes to which Psammotermes is presumed to be phylogenetically close. Our findings underline once again the conservative nature of chemical communication in termites, with dodecatrienol being a frequent component of pheromonal signals in trail following and sex attraction and, at the same time, a tight evolutionary relationship between the trail following of working castes and the sex attraction of imagos.

In the present study, trail pheromone blends are identified for the first time in termites. In the phylogenetically complex Nasutitermitinae, trail-following pheromones are composed of dodecatrienol and neocembrene, the proportions of which vary according to species, although neocembrene is always more abundant than dodecatrienol (by 25–250-fold). Depending on species, termites were more sensitive to dodecatrienol or to neocembrene but the association of both components always elicited significantly higher trail following, with a clear synergistic effect in most of the studied species. A third component, trinervitatriene, was identified in the sternal gland secretion of several species, but its function remains unknown. The secretion of trail pheromone blends appears to be an important step in the evolution of chemical communication in termites. The pheromone optimizes foraging, and promotes their ecological success.

Two mass spectrometric methods for analysing triacylglycerols (HPLC/APCI-MS and MALDI-MS) were used and compared in terms of the relevance of the data for further biostatistical evaluation. While MALDI-MS is simpler and significantly faster, the time-consuming and labour-intensive HPLC/APCI-MS provides more complete information about the lipid components. However, both methods provide well-comparable results concerning the grouping of specimens belonging to different species when evaluated with multivariate exploratory approaches. The compositions of triacylglycerols in the fat bodies of males in 11 bumblebee species (Bombus terrestris, B. lucorum, B. lapidarius, B. pratorum, B. sylvarum, B. ruderatus, B. pomorum, B. subterraneus, B. campestris, B. bohemicus, and B. rupestris) were found to be species-specific.

Although the frontal gland has long been known as a prominent defensive device for termite soldiers in many Rhinotermitidae and Termitidae, almost nothing is known about its function in imagoes. In the present study, we show that the frontal gland of imagoes in Prorhinotermes species is well developed at the time of the nuptial flight, and is filled with a complex mixture of sesquiterpene hydrocarbons and nitroalkenes. The sesquiterpene composition varies between Prorhinotermes simplex and Prorhinotermes canalifrons, between geographically distant colonies of P. simplex (Cuba versus Florida), and even between different flights of closely-related subcolonies. The ratio between (E)-1-nitropentadec-1-ene and sesquiterpenes is sex-specific. The volume of secretory cells decreases in functional kings and queens after colony foundation, and the subcellular organization changes into a form resembling unmodified epidermal cells. Dealate reproductives lose the ability for biosynthesis, and their frontal gland is devoid of volatile compounds found in swarming imagoes. The results obtained in the present study clearly show that the frontal gland is only temporarily active at the time of the dispersal flight. The most likely function of this gland is defence by the toxic nitroalkenes. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 384–392.

Unsymmetrically 3,3'-substituted axially chiral bis(tetrahydroisoquinoline) N,N'-dioxides can be prepared in just three steps. They exhibit unique catalytic activity (turnover frequency, enantioselectivity, substrate scope) in the asymmetric allylation of aromatic aldehydes (up to 96% ee). The product of the enantioselective allylation of benzaldehyde served as a building block for the preparation of an intermediate useful in the enantioselective synthesis of diospongines.

An HPLC non-aqueous reversed-phase separation system was adapted for analyzing insect triacylglycerols (TAG). The method uses two conventional Nova-Pak C18 columns connected in series, for a total length of 45 cm. The mobile phase gradient is mixed from acetonitrile and 2-propanol, and the flow rate is programmed from 1.0 to 0.7 mL/min. TAG are detected by atmospheric pressure chemical ionizationmass spectrometry. The method ensures efficient separation of isomers and analysis of high-molecularweight TAG with equivalent chain lengths up to 72. The method performance is demonstrated on analysis of TAG isolated from the fat body of the bumblebee Bombus lucorum.

A detailed analysis of Rhaponticum carthamoides (Willd.) Iljin root essential oil was carried out by GC, GC–MS and GC–FTIR techniques. In total, 30 components were identified, accounting for 98.0% of total volatiles. A norsesquiterpene 13-norcypera-1(5),11(12)-diene (22.6%), followed by aplotaxene (21.2%) and cyperene (17.9%), were isolated and their structures confirmed by 1D and 2D-NMR spectra (COSY, ROESY, HSQC, HMBC and INADEQUATE). Selinene type sesquiterpenes and aliphatic hydrocarbons were among minor constituents of the essential oil. The oil exhibited antimicrobial activity against 5 of 9 strains of bacteria and yeast, when tested using broth micro-dilution method. Minimum inhibitory concentrations ranged between 32 and 256 μg/ml.

Nigella sativa L. seed essential oils obtained by hydrodistillation (HD), dry steam distillation (SD), steam distillation of crude oils obtained by solvent extraction (SE-SD), and supercritical fluid extraction (SFE-SD) were tested for their antibacterial activities, using the broth microdilution method and subsequently analyzed by gas chromatography and gas chromatography-mass spectrometry. The results showed that the essential oils tested differed markedly in their chemical compositions and antimicrobial activities. The oils obtained by HD and SD were dominated by p-cymene, whereas the major constituent identified in both volatile fractions obtained by SD of extracted oils was thymoquinone (ranging between 0.36 and 0.38 g/ml, whereas in oils obtained by HD and SD, it constituted only 0.03 and 0.05 g/ml, respectively). Both oils distilled directly from seeds showed lower antimicrobial activity (MICs > or = 256 and 32 microg/ml for HD and SD, respectively) than those obtained by SE-SD and SFE-SD (MICs > or = 4 microg/ml). All oil samples were significantly more active against gram-positive than against gram-negative bacteria. Thymoquinone exhibited potent growth-inhibiting activity against gram-positive bacteria, with MICs ranging from 8 to 64 microg/ml.

A new class of axially chiral bipyridine N,N¢-oxides with bis(tetrahydroisoquinoline) framework were tested as catalysts in the reaction of aliphatic aldehydes with allyl(trichloro)silane to af- ford homoallylic alcohols. The course of the reaction, that is, the catalytic activity as well as enantioselectivity, is strongly dependent on the solvent used. The products were obtained in good yields and up to 68% ee in chloroform.