Sphinganine-like biogenesis of (E)-1-nitropentadec-1-ene in termite soldiers of the genus Prorhinotermes.

In 1974, (E)-1-nitropentadec-1-ene, a strong lipophilic contact poison of soldiers of the termite genus Prorhinotermes, was the first-described insect-produced nitro compound. However, its biosynthesis remained unknown. In the present study, we tested the hypothesis that (E)-1-nitropentadec-1-ene biosynthesis originates with condensation of amino acids with tetradecanoic acid. By using in vivo experiments with radiolabeled and deuterium-labeled putative precursors, we show that (E)-1-nitropentadec-1-ene is synthesized by the soldiers from glycine or L-serine and tetradecanoic acid. We propose and discuss three possible biosynthetic pathways.

Total synthesis, proof of absolute configuration, and biosynthetic origin of stylopsal, the first isolated sex pheromone of strepsiptera.

The asymmetric total synthesis of the diastereomers of stylopsal establishes the absolute configuration of the first reported sex pheromone of the twisted-wing parasite Stylops muelleri as (3R,5R,9R)-trimethyldodecanal. The key steps for the diastereo- and enantiodivergent introduction of the methyl groups are two different types of asymmetric conjugate addition reactions of organocopper reagents to α,ß-unsaturated esters, whereas the dodecanal skeleton is assembled by Wittig reactions. The structure of the natural product was confirmed by chiral gas chromatography (GC) techniques, GC/MS and GC/electroantennography (EAD) as well as field tests. An investigation into the biosynthesis of the pheromone revealed that it is likely to be produced by decarboxylation of a 4,6,10-trimethyltridecanoic acid derivative, which was found in substantial amounts in the fat body of the female, but not in the host bee Andrena vaga. This triple-branched fatty acid precursor thus seems to be biosynthesized de novo through a polyketide pathway with two consecutive propionate-propionate-acetate assemblies to form the complete skeleton. The simplified, motionless and fully host-dependent female exploits a remarkable strategy to maximize its reproductive success by employing a relatively complex and potent sex pheromone.

Nonadecadienone, a new termite trail-following pheromone identified in Glossotermes oculatus (Serritermitidae).

Within the multitude of chemical signals used by termites, the trail marking by means of pheromones is ubiquitous. Chemistry and biology of the trail-following communication have been described in more than 60 species from all families except for the Neotropical Serritermitidae. The chemical ecology of Serritermitidae is of special interest not only as a missing piece of knowledge on the diversity and evolution of isopteran pheromones but also because it may contribute to the debate on the phylogenetic position of this family, which is still unresolved. Therefore, we aimed in this study to identify the trail-following pheromone of the serritermitid Glossotermes oculatus. Based on a combined approach of analytical chemistry, electrophysiology, and behavioral bioassays, we propose (10Z,13Z)-nonadeca-10,13-dien-2-one to be the trail-following pheromone of G. oculatus, secreted by the sternal gland of pseudergates. Thus, we report on a new termite trail-following pheromone of an unexpected chemical structure, a ketone with 19 carbons, contrasting with unsaturated alcohols containing 12 carbons as trail-following pheromones in other advanced termite families. In addition to this unique trail-following pheromone, we also describe the sternal gland in pseudergates as an organ of unusual shape, size, and structure when compared with other isopteran species. These results underline the peculiarity of the family Serritermitidae and prompt our interest in the chemistry of pheromones in the other genus of the family, Serritermes.

Selective olfactory attention of a specialised predator to intraspecific chemical signals of its prey.

Prey-specialised predators have evolved specific cognitive adaptations that increase their prey searching efficiency. In particular, when the prey is social, selection probably favours the use of prey intraspecific chemical signals by predatory arthropods. Using a specialised ant-eating zodariid spider, Zodarion rubidum, which is known to prey on several ant species and possesses capture and venom adaptations more effective on Formicinae ants, we tested its ability to recognise chemical cues produced by several ant species. Using an olfactometer, we tested the response of Z. rubidum towards air with chemical cues from six different ant species: Camponotus ligniperda, Lasius platythorax and Formica rufibarbis (all Formicinae); and Messor structor, Myrmica scabrinodis and Tetramorium caespitum (all Myrmicinae). Z. rubidum was attracted to air carrying chemical cues only from F. rufibarbis and L. platythorax. Then, we identified that the spiders were attracted to airborne cues coming from the F. rufibarbis gaster and Dufour's gland, in particular. Finally, we found that among several synthetic blends, the decyl acetate and undecane mixture produced significant attraction of spiders. These chemicals are produced only by three Formicine genera. Furthermore, we investigated the role of these chemical cues in the communication of F. rufibarbis and found that this blend reduces their movement. This study demonstrates the chemical cognitive capacity of Z. rubidum to locate its ant prey using chemical signals produced by the ants. The innate capacity of Z. rubidum to olfactory detect different ant species is narrow, as it includes only two ant genera, confirming trophic specialisation at lower than subfamily level. The olfactory cue detected by Zodarion spiders is probably a component of the recruitment or trail pheromone.

Leg tendon glands in male bumblebees (Bombus terrestris): structure, secretion chemistry, and possible functions.

Among the large number of exocrine glands described in bees, the tarsal glands were thought to be the source of footprint scent marks. However, recent studies showed that the compounds used for marking by stingless bees are secreted by leg tendon instead of tarsal glands. Here, we report on the structure of leg tendon glands in males of Bombus terrestris, together with a description of the chemical composition of their secretions and respective changes of both during the males' lives. The ultrastructure of leg tendon glands shows that the secretory cells are located in three independent regions, separated from each other by unmodified epidermal cells: in the femur, tibia, and basitarsus. Due to the common site of secretion release, the organ is considered a single secretory gland. The secretion of the leg tendon glands of B. terrestris males differs in its composition from those of workers and queens, in particular by (1) having larger proportions of compounds with longer chain lengths, which we identified as wax esters; and (2) by the lack of certain hydrocarbons (especially long chain dienes). Other differences consist in the distribution of double bond positions in the unsaturated hydrocarbons that are predominantly located at position 9 in males but distributed at seven to nine different positions in the female castes. Double bond positions may change chemical and physical properties of a molecule, which can be recognized by the insects and, thus, may serve to convey specific information. The function of male-specific compounds identified from their tendon glands remains elusive, but several possibilities are discussed.

Females of the bumblebee parasite, Aphomia sociella, excite males using a courtship pheromone.

Aphomia sociella (Lepidoptera: Pyralidae: Galleriinae) is a parasitic moth of bumblebees. Behavioral experiments show that A. sociella females emit semiochemicals that influence male pre-mating behavior and serve as a courtship pheromone. GC/EAD and two-dimensional GC/MS (GCxGC-TOFMS) analyses of extracts of females revealed three antennally active compounds. Comparative GC and GCxGC-TOFMS analyses of extracts and synthetic standards confirmed the identity of the antennally active compounds as hexan-1-ol (1), 6,10,14-trimethylpentadecan-2-one (2), and 6,10,14-trimethylpentadecan-2-ol (3). In laboratory bioassays, alcohol 3 and, at higher doses, ketone 2 initiated male courtship behavior associated with ultrasonic production. Hexan-1-ol (1) and ketone 2 enhanced the activity of alcohol 3. These data suggest that hexan-1-ol, 6,10,14-trimethylpentadecan-2-ol, and 6,10,14-trimethylpentadecan-2-one constitute the female-produced courtship pheromone of A. sociella.

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

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

Essential oils in the ranunculaceae family: chemical composition of hydrodistilled oils from Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum seeds.

In this study, we analyzed the chemical composition of volatile oils hydrodistilled from seeds of Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum using GC and GC/MS. In C. regalis, octadecenoic (77.79%) and hexadecanoic acid (8.34%) were the main constituents. Similarly, the oils from D. elatum and N. hispanica seeds consisted chiefly of octadecadienoic (42.83 and 35.58%, resp.), hexadecanoic (23.87 and 28.59%, resp.), and octadecenoic acid (21.67 and 19.76%, resp.). Contrastingly, the monoterpene hydrocarbons α-pinene (34.67%) and ß-pinene (36.42%) were the main components of N. nigellastrum essential oil. Our results confirm the presence of essential oils in the family Ranunculaceae and suggest chemotaxonomical relationships within the representatives of the genera Consolida, Delphinium, and Nigella. In addition, the presence of various bioactive constituents such as linoleic acid, (-)-ß-pinene, squalene, or carotol in seeds of D. elatum, N. hispanica, and N. nigellastrum indicates a possible industrial use of these plants.

Sex pheromone and trail pheromone of the sand termite Psammotermes hybostoma.

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.

Composition and electrophysiological activity of constituents identified in male wing gland secretion of the bumblebee parasite Aphomia sociella.

Male wing gland secretion and volatiles emanating from calling males were investigated in the bumble bee wax moth, Aphomia. sociella, using comprehensive two-dimensional gas chromatography-mass spectrometry (GCxGC-TOF-MS), gas chromatography-infrared spectroscopy (GC-FTIR), enantioselective gas chromatography, electroantennography (EAG), gas chromatography with electroantennographic detection (GC-EAD), and NMR. GC-EAD analysis of the male wing gland secretion revealed seven active areas, corresponding to 1-hexanol (1), 2-phenylethanol (2), [(R),(Z)]-nona-2,6-dien-4-olide (3), [(S),(Z)]-nona-6-en-4-olide (4), mellein (5), phytone (6), and a mixture of C(18) fatty acids (7). Solid-phase microextraction (SPME) confirmed the presence of 2-phenylethanol, nona-2,6-dien-4-olide, nona-6-en-4-olide, mellein, and phytone in volatiles emanating from a calling male. Though the abundance of these compounds slightly differed in SPME and gland secretion analysis, nona-2,6-dien-4-olide and mellein dominated in both samples, followed by 2-phenylethanol, nona-6-en-4-olide, and phytone. The strong antennal responses elicited by components of the secretion suggest that one or more of these compounds constitute the sex pheromone. Both sexes perceived male wing gland secretion, with females being significantly more sensitive compared to males.

A delta9 desaturase from Bombus lucorum males: investigation of the biosynthetic pathway of marking pheromones.

The knowledge of the molecular basis of communication in bumblebee communities is limited. None of the enzymes that participate in pheromone production have been characterized. Here, we cloned the gene encoding the Delta(9) desaturase from cDNA prepared from the total RNA of the pheromone gland and fat bodies of Bombus lucorum male. Functional expression of BlucNPVE desaturase in Saccharomyces cerevisiae and GC-MS analyses revealed its preference for C(18) saturated fatty acids. This suggests that Delta(9) desaturase is involved in the desaturation of metabolic fatty acids stored in triacylglyceroles (TAGs), because oleic acid is the most abundant fatty acid bound in TAG in B. lucorum and it is present in low concentration in the pheromone blend. The incubation of pheromone precursors with a dissected labial gland as well as direct injection of labelled pheromone substrates into B. lucorum males revealed that esterification of pheromone products occurs in the labial gland. These results support both the biosynthesis of pheromones from common lipids and the de novo synthesis of unsaturated pheromones in the labial gland.

Unusual fatty acids in the fat body of the early nesting bumblebee, Bombus pratorum.

Unusual fatty acids with 24, 26, and 28 carbon atoms were found in triacylglycerols (TAGs) isolated from fat body tissue of bumblebee Bombus pratorum. The most abundant one was (Z,Z)-9,19-hexacosadienoic acid. Its structure was determined by mass spectrometry after derivatization with dimethyl disulfide and by infrared spectroscopy. ECL (equivalent chain length) values of its methyl ester were determined on both DB-1 and DB-WAX capillary columns. (Z,Z)-9,19-Hexacosadienoic acid is quite rare in nature. So far it has been identified only in marine sponges, and this work is the first evidence of its occurrence in a terrestrial organism. HPLC/MS analysis of the bumblebee TAGs showed that (Z,Z)-9,19-hexacosadienoic acid is present in one third of all TAG molecular species. As it was found in all sn-TAG positions, it is likely that (Z,Z)-9,19-hexacosadienoic acid is transported to tissues. Interestingly, labial gland secretion of B. pratorum was found to contain (Z,Z)-7,17-pentacosadiene, a hydrocarbon with markedly similar double bond positions and geometry. Possible biosynthetic relationships between these two compounds are discussed.

Analysis of triacylglycerols in fat body of bumblebees by chromatographic methods.

Triacylglycerols (TAGs) from the fat body of several bumblebee species (Bombus lucorum, B. terrestris, B. lapidarius, B. hypnorum, B. hortorum, and B. confusus) were studied using chromatographic techniques. Semi-preparative thin-layer chromatography was used to isolate the TAGs from the tissue extract. Gas chromatography (GC) enabled us to identify the fatty acids (FAs) that form bumblebee TAGs and to quantify their relative proportions. The TAGs were subsequently analysed by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry. Two chromatographic systems, including non-aqueous reversed-phase chromatography and silver ion chromatography on cation exchange resin in silver (I) ionic form, were optimised and their performance compared. The most abundant fatty acids in bumblebees TAGs contained 18 or 16 carbon atoms; oleic acid predominated in most samples. TAGs were found to be a complex mixture of isomers; some of them, e.g. OLnO, PLnO, PoPoO, PoPoP, POO, or OOO (where Po is palmitoleic, P is palmitic, Ln is linolenic, and O is oleic acid) were abundant in particular species. The composition of both FAs and TAGs was found to be species-specific. Only minor differences were found among specimens of the same species.

Seasonal Dynamics in the Chemistry and Structure of the Fat Bodies of Bumblebee Queens.

Insects' fat bodies are responsible for nutrient storage and for a significant part of intermediary metabolism. Thus, it can be expected that the structure and content of the fat body will adaptively change, if an insect is going through different life stages. Bumblebee queens belong to such insects as they dramatically change their physiology several times over their lives in relation to their solitary overwintering, independent colony foundation stage, and during the colony life-cycle ending in the senescent stage. Here, we report on changes in the ultrastructure and lipid composition of the peripheral fat body of Bombus terrestris queens in relation to seasonal changes in the queens' activity. Six life stages are defined and evaluated in particular: pharate, callow, before and after hibernation, egg-laying, and senescence. Transmission electron microscopy revealed that the fat body contained two main cell types-adipocytes and oenocytes. Only adipocytes reveal important changes related to the life phase, and mostly the ration between inclusion and cytoplasm volume varies among particular stages. Both electron microscopy and chemical analyses of lipids highlighted seasonal variability in the quantity of the stored lipids, which peaked prior to hibernation. Triacylglycerols appeared to be the main energy source during hibernation, while the amount of glycogen before and after hibernation remained unchanged. In addition, we observed that the representation of some fatty acids within the triacylglycerols change during the queen's life. Last but not least, we show that fat body cell membranes do not undergo substantial changes concerning phospholipid composition in relation to overwintering. This finding supports the hypothesis that the cold-adaptation strategy of bumblebee queens is more likely to be based on polyol accumulation than on the restructuring of lipid membranes.

Corylucinine, a new alkaloid from Corydalis cava (Fumariaceae), and its cholinesterase activity.

A new benzyldihydroisoquinoline alkaloid (1) was isolated from the tubers of Corydalis cava and named corylucinine. Additionally, 8-trichloromethyl-7,8-dihydropalmatine (2), an isolation artifact of tetrahydropalmatine, was obtained. The structures were established by spectroscopic (including 2D NMR and optical rotation) and HR-ESI-MS methods. Both compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. In comparison with the used standards, both compounds showed only moderate inhibitory activity against HuAChE (IC50,. HuAChE = 127.6 +/- 5.2 microM for 1, and IC50, HuAChE = 82.9 +/- 3.9 microM for 2) and none against HuBuChE.

Developmental pathways of Psammotermes hybostoma (Isoptera: Rhinotermitidae): old pseudergates make up a new sterile caste.

BACKGROUND: Ergonomic efficiency is in termites maximized by task partitioning among specialized castes. The isopteran caste systems can be classified as either (i) linear, when tasks are performed by pluripotent immatures (pseudergates), retaining the ability to develop into winged imagoes or (ii) bifurcated, with the presence of a true worker caste, which diverges early and permanently from the sexual (nymph/alate) line. PRINCIPAL FINDINGS: Here, we report on the ontogenetic potentialities of the highly polymorphic sand termite Psammotermes hybostoma. Beside numerous pluripotent pseudergates, constituting the main work force, some larger non-feeding apterous immatures, also occur. These individuals are unable to proceed to the winged imago stage, but store large amounts of fat and also give rise to large soldiers. Soldiers therefore originate from a wide range of apterous instars, consequently being highly polymorphic. CONCLUSIONS: The caste system of P. hybostoma is essentially linear, as in other basal Rhinotermitidae, but is distinguished by the late bifurcation leading to large apterous immatures. Because these large worker-like individuals deviate late and do not perform worker tasks, they cannot be considered homologous to the true workers of Termitidae and advanced Rhinotermitidae, but they provide a novel example of the evolution of sterile immatures in termites.

Changes in the composition of triacylglycerols in the fat bodies of bumblebee males during their lifetime.

The age-dependent changes in the composition of triacylglycerols (TAG) in the fat bodies of bumblebee males were studied using HPLC/MS. Two related species (Bombus terrestris and B. lucorum) were compared, with the age of the males being 0-30 days. The total amount of TAG in B. lucorum was about 2.7 times higher than that in B. terrestris for all of the ages studied. One to three-day-old males had the highest content of TAG in their fat bodies (1.6-2.3 mg/individual in B. terrestris and 3.8-4.2 mg/individual in B. lucorum). The analytical data show different patterns in both species. The qualitative composition of fatty acids in TAG was similar, but the mean relative abundance between B. terrestris and B. lucorum differed: 14:0, 7 and 14%; 16:0, 20 and 44%; 18:3, 62 and 23%; 18:1, 3 and 8%, respectively (the data is based on a GC/MS integration). A statistical evaluation of the dynamic changes in the TAG composition revealed that in B. terrestris different age classes were well separated according to their TAG composition while in B. lucorum the TAG did not change substantially during the male's life. The TAG analyses provide more precise information on the differences between the classes studied than the FA composition alone.

Analysis of Amaryllidaceae alkaloids from Chlidanthus fragrans by GC-MS and their cholinesterase activity.

The underivatized alkaloid mixture extracted from the bulbs of Chlidanthus fragrans Herb. was investigated by capillary GC/MS for the first time. Fifteen known Amaryllidaceae alkaloids of five structure types were identified. The main alkaloids were tazzetine (9, tazettine-type), chlidanthine (2, galanthamine-type), belladine (8, belladine-type) and lycorine (12, lycorine-type). The alkaloid extract from the bulbs showed promising human blood acetylcholinesterase (IC50 = 20.1 +/- 2.9 microg/mL) and human plasma butyrylcholinesterase (IC50 = 136.8 +/- 6.9 microg/mL) inhibitory activity.

Identification by GC-EAD of the two-component trail-following pheromone of Prorhinotermes simplex (Isoptera, Rhinotermitidae, Prorhinotermitinae).

GC/MS analysis confirmed that neocembrene is the major component of the trail pheromone in the three species of the termite genus Prorhinotermes (P. simplex, P. canalifrons, P. inopinatus). In addition, EAG and GC-EAD experiments with P. simplex strongly suggest that dodecatrienol is a quantitatively minor component but a qualitatively important component of this trail pheromone. Trail-following bioassays confirmed the two-component nature of the trail pheromone. This is the first report of the use of the GC-EAD for the identification of trail pheromone in termites. These original results underline once again the special phylogenetic status of the Prorhinotermitinae among Rhinotermitidae.

A comparison of HPLC/APCI-MS and MALDI-MS for characterising triacylglycerols in insects: species-specific composition of lipids in the fat bodies of bumblebee males.

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.