Novel, male-produced aggregation pheromone of the cerambycid beetle Rosalia alpina, a priority species of European conservation concern.

Several recent studies have demonstrated the great potential for exploiting semiochemicals in ecology and conservation studies. The cerambycid beetle Rosalia alpina represents one of the flagship species of saproxylic insect biodiversity in Europe. In recent years its populations appear to have declined substantially, and its range has shrunk considerably as a result of forest management and urbanization. Here, we collected volatile chemicals released by males and females of R. alpina. Analyses of the resulting extracts revealed the presence of a single male-specific compound, identified as a novel alkylated pyrone structure. In field bioassays in Slovenia, traps baited with the synthesized pyrone captured both sexes of R. alpina, indicating that the pyrone functions as an aggregation pheromone. Our results represent the first example of a new structural class of pheromones within the Cerambycidae, and demonstrate that pheromone-baited traps can provide a useful tool for sampling R. alpina. This tool could be particularly useful in the ongoing development of conservation strategies for the iconic but endangered Alpine longicorn.

Cholesteryl esters of ω-(O-acyl)-hydroxy fatty acids in vernix caseosa.

Cholesteryl esters of ω-(O-acyl)-hydroxy FAs (Chl-ωOAHFAs) were identified for the first time in vernix caseosa and characterized using chromatography and MS. Chl-ωOAHFAs were isolated using adsorption chromatography on silica gel and magnesium hydroxide. Their general structure was established using high-resolution and tandem MS of intact lipids, and products of their transesterification and derivatizations. Individual molecular species were characterized using nonaqueous reversed-phase HPLC coupled to atmospheric pressure chemical ionization. The analytes were detected as protonated molecules, and their structures were elucidated in the negative ion mode using controlled thermal decomposition and data-dependent fragmentation. About three hundred molecular species of Chl-ωOAHFAs were identified in this way. The most abundant Chl-ωOAHFAs contained 32:1 ω-hydroxy FA (ω-HFA) and 14:0, 15:0, 16:0, 16:1, and 18:1 FAs. The double bond in the 32:1 ω-HFA was in the n-7 and n-9 positions. Chl-ωOAHFAs are estimated to account for approximately 1-2% of vernix caseosa lipids.

Analysis of 1,2-diol diesters in vernix caseosa by high-performance liquid chromatography - atmospheric pressure chemical ionization mass spectrometry.

Fatty acid diesters of long-chain 1,2-diols (1,2-DDE), or type II wax diesters, were analyzed in the vernix caseosa of a newborn girl. 1,2-DDE were isolated from the total lipid extract by the semipreparative TLC using plates coated with silica gel. Chromatographic separation of the 1,2-DDE molecular species was achieved on the non-aqueous reversed-phase HPLC with two Nova-Pak C18 columns connected in series (a total length of 45cm) and using an acetonitrile-ethyl acetate gradient. 1,2-DDE eluted from the column in the order of their equivalent chain number. The analytes were detected as ammonium adducts by an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionization source. Their structures were elucidated using tandem mass spectrometry with MS, MS(2) and MS(3) steps in a data-dependent mode. More than two thousand molecular species of 1,2-DDE were identified in 141 chromatographic peaks. The most abundant 1,2-DDE were monounsaturated lipids consisting of a C22 diol and a C18:1 fatty acid together with C16:0, C14:0 or C15:0 fatty acids. The positions of double bonds were characterized by the fragmentation of [M+C3H5N](+) formed in the ion source.

Analyses of volatiles produced by the African fruit fly species complex (Diptera, Tephritidae).

Ceratitis fasciventris, Ceratitis anonae and Ceratitis rosa are polyphagous agricultural pests originating from the African continent. The taxonomy of this group (the so-called Ceratitis FAR complex) is unclear. To clarify the taxonomic relationships, male and female-produced volatiles presumably involved in pre-mating communication were studied using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) followed by multivariate analysis, and gas chromatography combined with electroantennographic detection (GC-EAD). GC×GC-TOFMS analyses revealed sex specific differences in produced volatiles. Male volatiles are complex mixtures that differ both qualitatively and quantitatively but share some common compounds. GC-EAD analyses of male volatiles revealed that the antennal sensitivities of females significantly differ in the studied species. No female volatiles elicited antennal responses in males. The results show clear species-specific differences in volatile production and provide complementary information for the distinct delimitation of the putative species by chemotaxonomic markers.

Cuticular hydrocarbons of the South American fruit fly Anastrepha fraterculus: variability with sex and age.

Insect cuticular hydrocarbons are usually species-specific mixtures and may serve for species and gender recognition. They are, therefore, widely used in the chemotaxonomy and zoogeography of various insect taxa. In order to provide a basic study for further comparative analyses of cuticular hydrocarbon (CHC) profiles of cryptic species hidden within the South American fruit fly Anastrepha fraterculus complex (Diptera: Tephritidae), we analyzed the composition of the CHCs and their production with respect to age and sex in a laboratory population from Tucuman, Argentina. Several techniques of gas chromatography with mass spectrometric detection have been used in order to develop a suitable method for CHC identification, i.e., GC-MS in EI mode, GC-MS in CI mode, and GC×GC/TOFMS. Our analyses revealed a complex profile of aliphatic hydrocarbons in both males and females, consisting predominantly of n-alkanes, methyl-branched alkanes, as well as of alkenes and alkadienes. In young individuals (up to about 5 days after emergence), the CHC profiles were similar in males and females. However, in older flies, these profiles diverged and became clearly sex-specific. The temporal dynamics of the CHC patterns in both sexes were evaluated using multivariate exploratory techniques.

Are the wild and laboratory insect populations different in semiochemical emission? The case of the medfly sex pheromone.

The medfly (Ceratitis capitata) is one of the major agricultural pests controlled through sterile insect technique (SIT) programs. We studied the chemical composition of the volatiles released by calling males from one laboratory and two wild C. capitata populations using two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC × GC/TOFMS) and gas chromatography with electroantennographic detection (GC-EAD). Multivariate data analyses revealed significant differences in the quantitative and qualitative composition of male chemical emanations between the three populations. The GC-EAD analyses of the male emanation of three C. capitata populations revealed 14 antenally active compounds. The volatiles isomenthone, ß-pinene, ethyl octanoate, indole, geraniol, bornyl acetate, geranyl acetone, and (E)-caryophyllene are newly reported EAD active constituents of the male pheromone. GC-EAD analyses of the laboratory population indicated that the males and females of C. capitata possess comparable sensitivity to male-produced volatiles. Our results are relevant to the development of a pheromone-based monitoring system and also to the SIT control program.

Chemical communication in termites: syn-4,6-dimethylundecan-1-ol as trail-following pheromone, syn-4,6-dimethylundecanal and (5E)-2,6,10-trimethylundeca-5,9-dienal as the respective male and female sex pheromones in Hodotermopsis sjoestedti (Isoptera, Archotermopsidae).

The trail-following pheromone and sex pheromones were investigated in the Indomalayan termite Hodotermopsis sjoestedti belonging to the new family Archotermopsidae. Gas chromatography coupled to mass spectrometry (GC-MS) after solid phase microextraction (SPME) of the sternal gland secretion of pseudergates and trail-following bioassays demonstrated that the trail-following pheromone of H. sjoestedti was syn-4,6-dimethylundecan-1-ol, a new chemical structure for termite pheromones. GC-MS after SPME of the sternal gland secretion of alates also allowed the identification of sex-specific compounds. In female alates, the major sex-specific compound was identified as (5E)-2,6,10-trimethylundeca-5,9-dienal, a compound previously identified as the female sex pheromone of the termite Zootermopsis nevadensis. In male alates, the major sex-specific compound was identified as syn-4,6-dimethylundecanal, a homolog of syn-4,6-dimethyldodecanal, which has previously been confirmed as the male sex pheromone of Z. nevadensis. The presence of sex-specific compounds in alates of H. sjoestedti strongly suggests for this termite the presence of sex-specific pairing pheromones which were only known until now in Z. nevadensis. Our results showed therefore a close chemical relationship between the pheromones of the taxa Hodotermopsis and Zootermopsis and, in contrast, a clear difference with the taxa Stolotermes and Porotermes, which is in total agreement with the recent creation of the families Archotermopsidae and Stolotermitidae as a substitute for the former family Termopsidae.

GCxGC/TOF MS technique-A new tool in identification of insect pheromones: Analysis of the persimmon bark borer sex pheromone gland.

Conventional gas chromatography with electroantennographic detection (GC-EAD) and two-dimensional (GCxGC) gas chromatography using a time-of-flight mass spectrometric detector (TOFMS), were combined to analyse the female sex pheromone gland extract of the persimmon bark borer, Euzophera batangensis. GC-EAD analysis produced two EAD responses in GC areas where no compounds were detected by FID detection. GCxGC/TOFMS analysis of this area indicated the presence of several chemicals, including (Z9,E12)-tetradeca-9,12-dien-1-ol and (Z9)-tetradec-9-en-1-ol, pheromone components of closely related Euzophera species. Spectral characteristics, retention behaviour and the ability to elicit GC-EAD responses imply that both identified unsaturated alcohols are candidates for E. batangensis sex pheromone components. GCxGC/TOFMS facilitated the analysis of complex matrices on a subnanogram level and was shown to have great potential as a powerful tool in the analysis of insect pheromones.

Determining the vapour pressures of plant volatiles from gas chromatographic retention data.

The frequently used vapour pressure versus Kováts retention index relationship has been evaluated in terms of its universal applicability, highlighting the problems associated with predicting the vapour pressures of structurally divergent organic compounds from experimentally measured isothermal Kováts retention indices. Two models differing in approximations adopted to express the activity coefficient ratio have been evaluated using 32 plant volatiles of different structural types as a test set. The validity of these models was established by checking their ability to reproduce 22 vapour pressures known from independent measurements. Results of the comparison demonstrated that (i) the original model, based on the assumption of equal activity coefficients for the test and reference substances, led, as expected, to a poor correlation (r2 = 89.1% only), with significantly deviating polar compounds and (ii) the model showed significant improvement after incorporating a new empirical term related to vaporization entropy and boiling point. The addition of this term allowed more than 99% of the vapour pressure variance to be accounted for. The proposed model compares favourably with existing correlations, while having an added advantage of providing a convenient tool for vapour pressure determination of chemically divergent chemicals.