Host dependent iridoid glycoside sequestration patterns in Cionus hortulanus.

Weevils of the genus Cionus (Curculionidae, Mecininae) sequester the iridoid glycosides (IGs) aucubin and catalpol from their host plants Scrophularia or Verbascum (Scrophulariaceae). Cionus hortulanus is the only member of the genus that feeds on both plant genera. We previously showed that sequestration patterns in C. hortulanus depend on the local host. To investigate whether IG patterns are driven by their availability in the hosts or genetic differences between populations, we collected C. hortulanus from S. nodosa in the field and reared them either on S. nodosa or on V. nigrum. The differences in IG concentrations were specific for the host plant upon which the weevils developed. Similar to monophagous species of the Cionini, individuals from S. nodosa had more aucubin than catalpol and mirrored the concentrations of their host plants. Specimens from V. nigrum, on the other hand, had higher concentrations of aucubin and of catalpol than their host. On V. nigrum, the ratio of catalpol to aucubin differed significantly between plant and beetle samples due to much higher catalpol concentrations in the weevils. Our data thus contradict genetically fixed differences between populations living on either plant but rather document the host plants' influence on the beetles' metabolism.

Scents of adolescence: the maturation of the olfactory phenotype in a free-ranging mammal.

Olfaction is an important sensory modality for mate recognition in many mammal species. Odorants provide information about the health status, genotype, dominance status and/or reproductive status. How and when odor profiles change during sexual maturation is, however often unclear, particularly in free-ranging mammals. Here, we investigated whether the wing sac odorant of male greater sac-winged bats (Saccopteryx bilineata, Emballonuridae) differs between young and adults, and thus offers information about sexual maturity to potential mating partners. Using gas chromatography-mass spectrometry, we found differences in the odorants of young and adult males prior and during, but not after the mating period. The wing sac odorant of adult males consists of several substances, such as Pyrocoll, 2,6,10-trimethyl-3-oxo-6,10-dodecadienolide, and a so far unidentified substance; all being absent in the odor profiles of juveniles prior to the mating season. During the mating season, these substances are present in most of the juvenile odorants, but still at lower quantities compared to the wing sac odorants of adults. These results suggest that the wing sac odorant of males encodes information about age and/or sexual maturity. Although female S. bilineata start to reproduce at the age of half a year, most males of the same age postpone the sexual maturation of their olfactory phenotype until after the first mating season.

(1R,2S,6R)-2-Hydroxymethyl-2,6-dimethyl-3-oxabicyclo[4.2.0]octane, a new volatile released by males of the papaya borer Pseudopiazurus obesus (Col.: Curculionidae).

Sex-specific volatiles produced by males of the papaya beetle Pseudopiazurus obesus are (1R,2S)-grandisal (1), (1R,2S)-grandisol (2), and the new (1R,2S,6R)-2-hydroxymethyl-2,6-dimethyl-3-oxabicyclo[4.2.0]octane (3) termed papayanol.

Ex post glycoconjugation of phthalocyanines.

For the first time, fully fledged phthalocyanines (Pcs) were ex post glycoconjugated, that is, via 1,3-dipolar cycloaddition reaction. This divergent approach gains rapid access to a broad range of highly diverse Pcs bearing chemically sensitive substituents. This will be a breakthrough in generating structure-activity relationships (SAR) for the development of novel bioactive molecules.

Identification of sex pheromone components of the hessian fly, Mayetiola destructor.

Coupled gas chromatographic (GC)-electroantennographic detection (EAD) analyses of ovipositor extract of calling Hessian fly, Mayetiola destructor, females revealed that seven compounds elicited responses from male antennae. Four of the compounds-(2S)-tridec-2-yl acetate, (2S,10Z)-10-tridecen-2-yl acetate, (2S,10E)-10-tridecen-2-yl acetate, and (2S,10E)-10-tridecen-2-ol-were identified previously in female extracts. Two new EAD-active compounds, (2S,8Z,10E)-8,10-tridecadien-2-yl acetate and (2S,8E,10E)-8,10-tridecadien-2-yl acetate, were identified by GC-mass spectroscopy (MS) and the use of synthetic reference samples. In a Y-tube bioassay, a five-component blend (1 ng (2S)-tridec-2-yl acetate, 10 ng (2S,10E)-10-tridecen-2-yl acetate, 1 ng (2S,10E)-10-tridecen-2-ol, 1 ng (2S,8Z,10E)-8,10-tridecadien-2-yl acetate, and 1 ng (2S,8E,10E)-8,10-tridecadien-2-yl acetate) was as attractive to male Hessian flies as a similar amount of female extract (with respect to the main compound, (2S,10E)-10-tridecen-2-yl acetate). The five-component blend was more attractive to male flies than a three-component blend lacking the two dienes. Furthermore, the five-component blend was more attractive than a blend with the same compounds but that contained one tenth the concentration of (2S,8E,10E)-8,10-tridecadien-2-yl acetate (more accurately mimicking the ratios found in female extract). This suggests that the ratios emitted by females might deviate from those in gland extracts. In a field-trapping experiment, the five-component blend applied to polyethylene cap dispensers in a 100:10 microg ratio between the main component and each of the other blend components attracted a significant number of male Hessian flies. Also, a small-plot field test demonstrated the attractiveness of the five-component blend to male Hessian flies and suggests that this pheromone blend may be useful for monitoring and predicting Hessian fly outbreaks in agricultural systems.

Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling.

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Delta4,8-desaturated ceramide as substrates.

A possible role of sphingolipids in the aluminium resistance of yeast and maize.

The plasma membrane is most likely the major target for sensing of aluminium (Al), leading to inhibition of plant root-growth. As a result of high external Al, alterations in plasma membrane composition may be expected in order to maintain its properties. As sphingolipids are characteristic components of this membrane, their involvement in membrane adjustment to increased Al concentrations was investigated. Heterologous expression of a stereounselective long-chain base (LCB) (8E/Z)-desaturase from Arabidopsis thaliana, Brassica napus and Helianthus annuus in Saccharomyces cerevisiae improved the Al resistance of the transgenic yeast cells. This encouraged us to investigate whether Al affects the LCB composition, and whether genetic engineering of the LCB profile modifies the Al resistance of the Al-sensitive plant species maize (Zea mays, L.). Constitutive expression of the LCB (8E/Z)-desaturase from Arabidopsis thaliana in maize roots led to an 8- to 10-fold increase in (8E)-4-hydroxysphing-8-enine in total roots. Less marked but similar changes were observed in 3 mm root apices. Al treatment of the Al-sensitive maize cv Lixis resulted in a significant increase in the proportion of (8Z)-LCB and in the content of total LCBs in root tips, which was not observed in the Al-resistant cv ATP-Y. When root tips of transgenic plants were exposed to Al, only minor changes of both (8Z)- and (8E)-unsaturated LCBs as well as of the total LCB were observed. Al treatment of the wild type parental line H99 decreased the (8Z)-unsaturated LCBs and the total LCB content. Based on Al-induced callose production, a marker for Al sensitivity, the parental line H99 was as Al-resistant as cv ATP-Y, whereas the transgenic line became as sensitive as cv Lixis. Taken together, these data suggest that, in particular, the loss of the ability to down-regulate the proportion of (8Z)-unsaturated LCBs may be related to increased Al sensitivity.

Identification of the sex pheromone of the swede midge, Contarinia nasturtii.

Coupled gas chromatographic-electroantennographic detection analyses of ovipositor extracts of calling Contarinia nasturtii females revealed two compounds that elicited responses from antennae of male midges. Using synthetic reference samples, these components were identified by gas chromatography mass spectrometry and enantioselective GC as (2S,9S)-diacetoxyundecane and (2S,10S)-diacetoxyundecane. In addition, trace amounts of 2-acetoxyundecane were found in ovipositor extracts, and the (S)-enantiomer was synthesized. When tested in the wind tunnel, a blend of 5 ng (2S,9S)-diacetoxyundecane and 10 ng (2S,10S)-diacetoxyundecane (mimicking the ratio found in the extracts) did not attract any of the males tested, but when 0.1 ng (S)-2-acetoxyundecane was added to the blend, 86.8% of the males were attracted to the bait. Three-component blends with lower or higher relative concentrations than 1% of (S)-2-acetoxyundecane [relative to (2S,10S)-diacetoxyundecane] were less attractive. In a field trapping experiment with released laboratory-reared C. nasturtii adults, traps baited with 500:1000:10 ng of (2S,9S)-diacetoxyundecane/(2S,10S)-diacetoxyundecane/(S)-2-acetoxyundecane applied to rubber septa or dental cotton rolls were tested. Traps without dispensers were used as controls. All three treatments were tested at 20 and 50 cm above ground. The estimated recapture rate was 30-50%, and 81.9% of the recaptured males were caught in traps positioned at 20 cm above ground, and 88.4% in traps with dental cotton rolls as dispensers.

Catabolism of Mesamoll, a technical formulation of alkylsulfonic acid phenyl esters, by two strains of Rhodococcus rhodochrous.

The bacterial catabolism of a technical formulation of alkylsulfonic acid phenyl esters employed as a general purpose plasticizer, Mesamoll, was elucidated. Two strains of the genus Rhodococcus were found able to utilize this substrate mixture as sole source of carbon and energy. Growth experiments along with enzymatic measurements indicated that both strains utilized the phenol that was released from the corresponding alkylsulfonic acid phenyl ester-probably by enzymatic hydrolysis catalyzed by esterases-via the ortho-pathway. By GC/MS analysis it was demonstrated that those alkyl chain homologues with substituents present at or close to the end of the aliphatic backbone (i.e., 2-tetradecylsulfonic acid phenyl ester) are degraded, while those with substituents close to the center of the alkyl chain (i.e., 7-tetradecylsulfonic acid phenyl ester) are rather persistent.

Vittatalactone, a beta-lactone from the striped cucumber beetle, Acalymma vittatum.

A novel beta-lactone, vittatalactone (1), was isolated from collections of airborne volatile compounds from feeding male striped cucumber beetles, Acalymma vittatum. The structure of 1 was determined to be (3R,4R)-3-methyl-4-(1,3,5,7-tetramethyloctyl)oxetan-2-one by microderivatization, GC-MS, and NMR studies. The absolute configurations at C-2 and C-3 on the beta-lactone ring were assigned by use of the modified Mosher method, applied to the beta-hydroxy acid methyl ester resulting from methanolysis of 1. Biological activity of 1, possibly as an aggregation pheromone for A. vittatum, was indicated by electrophysiological studies using beetle antennae and by the production of 1 by feeding male, and not female, beetles.

Are glucocerebrosides the predominant sphingolipids in plant plasma membranes?

Long-chain sphingobases have been analyzed in various fractions prepared from different organs (leaf, root, storage tissue) from five dicotyledoneous plants (Arabidopsis thaliana, Brassica oleracea, Nicotiana tabacum, Pisum sativum, Spinacia oleracea). The resulting sphingobase profiles from cerebrosides and plasma membranes (PMs) show large qualitative and quantitative differences. Assuming that cerebrosides from all cellular membranes have similar sphingobase profiles, these data suggest that cerebrosides, considered to be characteristic glycolipids of plant PMs and specified by large proportions of sphingobases with an 8Z-double bond motif, do not represent the major sphingolipids of PMs. The fraction of unidentified complex sphingolipids, containing mainly 8E-phytosphingenine, exceeds the cerebroside proportion in PMs by several factors and may be as abundant as diacylglycerol-based phospholipids. These results are discussed with respect to the distribution of various lipids between the bilayer halves of plant PM.

Identification of the sex pheromone of the currant shoot borer Lampronia capitella.

Under an artificial light:dark cycle, females of Lampronia capitella were observed calling, with extended terminal abdominal segments, during the first 2 hr of the photoperiod. Extracts of terminal abdominal segments from females elicited large electroantennographic responses from male antennae. Gas chromatography with electroantennographic detection revealed three active peaks. Based on comparison of retention times and mass spectra of synthetic standards, these compounds were identified as (Z,Z)-9,11-tetradecadienol and the corresponding acetate and aldehyde. The electroantennographic activity of the four geometric isomers of all three compounds was investigated, and the respective (Z,Z)-isomer was found to be the most active in all cases. Aldehydes generally elicited larger antennal responses than alcohols, whereas acetates were the least active compounds. A subtractive trapping assay in the field, based on a 13:26:100 micrograms mixture of (Z,Z)-9,11-tetradecadienal, (Z,Z)-9,11-tetradecadienyl acetate, and (Z,Z)-9,11-tetradecadienol confirmed that all three compounds are pheromone components. Subtraction of (Z,Z)-9,11-tetradecadienol from the blend completely eliminated its attractiveness, whereas the other two-component blends showed reduced activity. This is the first pheromone identification from the monotrysian superfamily Incurvarioidea, confirming that the common pheromones among ditrysian moths (long-chain fatty acid derivatives comprising alcohols, acetates, and aldehydes with one or more double bonds) is not an autapomorphy of Ditrysia, but a synapomorphy of the more advanced heteroneuran lineages.

Polymer-bound reagents for the introduction of spacer-modified biotin labels.

We have developed a method for the chemoselective introduction of spacer modified biotin labels into unprotected multi-functional amines. A range of novel biotin spacer conjugates attached to a polymer-bound sulfonamide anchor was prepared using established amide bond forming procedures. After chemical transformation of the attachment site by alkylation, the resulting reactive species were utilized as N-selective polymer-supported biotinylation reagents. The labeled compounds, obtained in good to excellent yield and purity, are free of residual biotin and possess a custom tailored distance from the immobilization site being especially suited for the immobilization on streptavidin-functionalized dextran layers of surface plasmon resonance detector chips. In addition, derivatives displaying a phenyl group were synthesized in order to demonstrate the versatility of the procedure for the simultaneous introduction of spacer-modified biotin and a UV-light absorbing moiety. The formation of biotin sulfoxides in the presence of in situ generated peroxides was investigated and is discussed. Our results suggest that this derivatization technique is a useful addition to the existing biotin labeling protocols.

Cuticular hydrocarbons and aggression in the termite Macrotermes subhyalinus.

Cuticular hydrocarbons are among the prime candidates for nestmate recognition in social insects. We analyzed the variation of cuticular hydrocarbons in the termite species M. subhyalinus in West Africa (Comoë National Park) on a small spatial scale (< 1 km). We found considerable variation in the composition of cuticular hydrocarbons among colonies, with four distinct chemical phenotypes. Different phenotypes occurred within each of the four habitats. The difference between these phenotypes is primarily due to unsaturated compounds. A clear correlation between the difference of the hydrocarbon composition and the aggression between colonies was found. This correlation also holds in a multivariate analysis of genetic similarity (measured by AFLPs), morphometric distances (measured by Mahalanobis-distances). as well as geographic distances between colonies. In a more detailed analysis of the correlation between the composition of cuticular hydrocarbons and aggression, we found that no single compound is sufficient to explain variation in aggression between pairings of colonies. Thus, termites seem to use a bouquet of compounds. Multiple regression analysis suggested that many of these compounds are unsaturated hydrocarbons and, thus, may play a key role in colony recognition.

Sex pheromone of the leaf-miner Phyllonorycter platani: (Z10)-tetradecenyl acetate.

A female produced sex pheromone of the leaf-miner pest Phyllonorycter platani (Staudinger) (Lepidoptera: Gracillariidae) was identified as (Z10)-tetradecenyl acetate. Field trapping trials confirmed activity of this compound. The presence of the geometrical isomer (E10)-tetradecenyl acetate, a pheromone component of other Phyllonorycter species, reduced trap efficacy.

The chemistry of sexual deception in an orchid-wasp pollination system.

The "sexually deceptive" orchid Chiloglottis trapeziformis attracts males of its pollinator species, the thynnine wasp Neozeleboria cryptoides, by emitting a unique volatile compound, 2-ethyl-5-propylcyclohexan-1,3-dione, which is also produced by female wasps as a male-attracting sex pheromone.

Identification and quantitation of dinaphthylsulfones in particulate matter of the Elbe river, Germany. Part VI of organic compounds as contaminants of the Elbe river and its tributaries.

Gas chromatographic-mass spectrometric (GC/MS) analysis of particulate matter of the Elbe river and its tributaries Havel, Spree and Mulde revealed a group of three dinaphthylsulfone isomers as sedimentary and suspended particulate matter (SPM) contaminants. The mass spectra of dinaphthylsulfones are characterized by the molecular ion (m/z 318), and the naphthyl fragment ion m/z 127. Losses of HSO(2) and C(10)H(7)O from the molecular ion lead to different mass spectra for each isomer. The gas phase infrared spectra exhibit isomer specific bands in the spectral region between 900 and 700 wave numbers. A synthetic mixture of dinaphthylsulfones was used for isomer identification and the assignment of the gas chromatographic retention behaviour of the dinaphthylsulfone isomers. Quantitative GC/MS analysis of dinaphthylsulfones in 44 sediment and SPM samples provided comprehensive information on the overall distribution and distinct sources of dinaphthylsulfones in the Elbe river drainage system. The results indicate emissions of these compounds over prolonged times and their environmental stability in anaerobic sediments.

Female sex pheromone of Cameraria ohridella Desch. and Dim. (Lepidoptera: Gracillariidae): structure confirmation, synthesis and biological activity of (8E,10Z)-8,10-tetradecadienal and some analogues.

Mass spectrometric investigations confirmed the structure of the female produced sex pheromone of the horse-chestnut leafminer Cameraria ohridella Desch. and Dim. to be (8E,IOZ)-8,10-tetradecadienal. Pure samples, prepared in a straightforward synthesis, were highly attractive in field tests and proved to be suitable for monitoring of flight activities and population dynamics. In mixtures with the synthetic pheromone, analogues like 9-tridecynal and 7-dodecynyl formate were shown to reduce trap catches. In electroantennographic experiments, pheromone analogues were less active than the pheromone. 9-Tridecynal was the most EAG active analogue tested, followed by 7-dodecyn-1-yl formate and 7-undecyn-1-yl formate.

Antimalarial activity of n(6)-substituted adenosine derivatives (part 2).

We have investigated the in vitro antimalarial activity of a new series of adenosine derivatives. The results show that N(6)-(1-naphthylmethyl)-5'-deoxy-5'-(amido)adenosines as well as N(6)-(4-phenylbenzyl)-5'-deoxy-5'-(amido)adenosines display significant activity against the malaria-causing parasites, with the sterically demanding bisubstituted species reported being active in most cases in the low-micromolar range. The novel compounds with unusual substitution pattern were obtained applying an efficient convergent polymer-assisted solution-phase (cPASP) synthesis protocol. Thus, we were able to prepare a series of substituted derivatives in parallel that would have been difficult to synthesize by standard techniques. The scope and limitations of the synthetic methodology are discussed.

Identification and characterization of a sphingolipid delta 4-desaturase family.

Sphingolipids desaturated at the Delta4-position are important signaling molecules in many eukaryotic organisms, including mammals. In a bioinformatics approach, we now identified a new family of protein sequences from animals, plants, and fungi and characterized these sequences biochemically by expression in Saccharomyces cerevisiae. This resulted in the identification of the enzyme sphingolipid Delta4-desaturase (dihydroceramide desaturase) from Homo sapiens, Mus musculus, Drosophila melanogaster, and Candida albicans, in addition to a bifunctional sphingolipid Delta4-desaturase/C-4-hydroxylase from M. musculus. Among the sequences investigated are the Homo sapiens membrane lipid desaturase, the M. musculus degenerative spermatocyte, and the Drosophila melanogaster degenerative spermatocyte proteins. During spermatogenesis, but not oogenesis of des mutant flies, both cell cycle and spermatid differentiation are specifically blocked at the entry into the first meiotic division, leading to male sterility. This mutant phenotype can be restored to wild-type by complementation with a functional copy of the des gene (Endo, K., Akiyama, T., Kobayashi S., and Okada, M. (1996) Mol. Gen. Genet. 253, 157-165). These results suggest that Delta4-desaturated sphingolipids provide an early signal necessary to trigger the entry into both meiotic and spermatid differentiation pathways during Drosophila spermatogenesis.