Cyclopropane Hydrocarbons from the Springtail Vertagopus sarekensis─A New Class of Cuticular Lipids from Arthropods.

The epicuticle of insects is usually coated with a complex mixture of hydrocarbons, primarily straight-chain and methyl-branched alkanes and alkenes. We were interested in whether springtails (Collembola), a sister class of the insects, also use such compounds. We focused here on Vertagopus sarekensis, an abundant Isotomidae species in European high alpine regions, exhibiting coordinated group behavior and migration. This coordination, suggesting chemical communication, made the species interesting for our study on epicuticular hydrocarbons in springtails with different degrees of group behavior. We isolated a single hydrocarbon from its surface, which is the major epicuticular lipid. The structure was deduced by NMR analysis and GC/MS including derivatization. Total synthesis confirmed the structure as cis,cis-3,4,13,14-bismethylene-24-methyldotriacontane (4, sarekensane). The GC/MS analyses of some other cyclopropane hydrocarbons also synthesized showed the close similarity of both mass spectra and gas chromatographic retention indices of alkenes and cyclopropanes. Therefore, analyses of cuticular alkenes must be performed with appropriate derivatization to distinguish these two types of cuticular hydrocarbons. Sarekensane (4) is the first nonterpenoid cuticular hydrocarbon from Collembola that is biosynthesized via the fatty acid pathway, as are insect hydrocarbons, and contains unprecedented cyclopropane rings in the chain, not previously reported from arthropods.

Compounds from human odor induce attraction and landing in female yellow fever mosquitoes (Aedes aegypti).

The female Aedes aegypti mosquito is a vector of many human diseases such as yellow fever, dengue, and Zika. Transmission of these viruses occurs when an infected female mosquito locates a suitable human host, alights, and blood feeds. Aedes aegypti use human-emitted odors, as well as heat and visual cues, for host location. However, none of the previously identified human-produced compounds induce significant orientation and landing on a human host. Here we show that female yellow fever mosquitoes orient to and land on a mixture of compounds identified from human skin rubbings. Using odor collection, extraction, a two-choice, bioassay-guided fractionation, and chemical analysis, we identified mixtures of 2-ketoglutaric acid and L-lactic acid as landing attractants for female Ae. aegypti. The mixture of pyruvic acid and L-lactic acid were also found to be weakly attractive. Using ratio-response assays, we found that the attraction and alighting behaviors of the mosquitoes were directly related to the ratio of these compounds presented on the surface of the glass assay beads, suggesting that these compounds could mediate landing on a human host even at sub-nanogram dosages. The newly identified compounds fill a gap in our knowledge of odor-mediated attraction of Ae. aegypti and may lead to the development of new attractant-based mosquito control tactics.

Targeting Insect Olfaction in vivo and in vitro Using Functional Imaging.

Insects decode volatile chemical signals from its surrounding environment with the help of its olfactory system, in a fast and reliable manner for its survival. In order to accomplish this task, odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) in the fly's antenna process such odor information. In order to study such a sophisticated process, we require access to the sensory neurons to perform functional imaging. In this article, we present different preparations to monitor odor information processing in Drosophila melanogaster OSNs using functional imaging of their Ca2+ dynamics. First, we established an in vivo preparation to image specific OSN population expressing the fluorescent Ca2+ reporter GCaMP3 during OR activation with airborne odors. Next, we developed a method to extract and to embed OSNs in a silica hydrogel with OR activation by dissolved odors. The odor response dynamics under these different conditions was qualitatively similar which indicates that the reduction of complexity did not affect the concentration dependence of odor responses at OSN level.

Variations on a Theme: Two Structural Motifs Create Species-Specific Pheromone Channels for Multiple Species of South American Cerambycid Beetles.

We describe the identification, synthesis, and field-testing of aggregation-sex pheromones, or likely pheromone candidates, of seven species of South American cerambycid beetles in the subfamily Cerambycinae, of the tribes Eburiini and Neoibidionini. Analyses of extracts of volatiles released by adult males revealed that Eburodacrys dubitata White produce 11-methyltridecanal, whereas the males of Eburodacrys assimilis Gounelle, Eburodacrys flexuosa Gounelle, and Eburodacrys lenkoi Napp and Martins produce blends of this compound, along with its analog 10-methyldodecanal. In contrast, males of Compsibidion graphicum (Thomson) and Compsibidion sommeri (Thomson) produce blends of 10-methyldodecanal and its corresponding alcohol 10-methyldodecanol. The results from field bioassays with synthetic compounds showed that each species was specifically attracted to traps containing their reconstructed pheromone blend. However, E. assimilis was not trapped, possibly due to inhibition by non-natural enantiomers in the racemic test compounds. During the trials for the Compsibidion species, adults of another cerambycid species, Tetraopidion mucoriferum (Thomson), were captured in significant numbers in traps baited with 10-methyldodecanol, suggesting that this compound is a pheromone component for this species. This study demonstrates another case of conservation of pheromone structures within South American cerambycid species. It also highlights how blends of closely related structures, differing only in chain length or functional group, make the evolution of species-specific pheromone channels possible.

Specialized odorant receptors in social insects that detect cuticular hydrocarbon cues and candidate pheromones.

Eusocial insects use cuticular hydrocarbons as components of pheromones that mediate social behaviours, such as caste and nestmate recognition, and regulation of reproduction. In ants such as Harpegnathos saltator, the queen produces a pheromone which suppresses the development of workers' ovaries and if she is removed, workers can transition to a reproductive state known as gamergate. Here we functionally characterize a subfamily of odorant receptors (Ors) with a nine-exon gene structure that have undergone a massive expansion in ants and other eusocial insects. We deorphanize 22 representative members and find they can detect cuticular hydrocarbons from different ant castes, with one (HsOr263) that responds strongly to gamergate extract and a candidate queen pheromone component. After systematic testing with a diverse panel of hydrocarbons, we find that most Harpegnathos saltator Ors are narrowly tuned, suggesting that several receptors must contribute to detection and discrimination of different cuticular hydrocarbons important in mediating eusocial behaviour.Cuticular hydrocarbons (CHC) mediate the interactions between individuals in eusocial insects, but the sensory receptors for CHCs are unclear. Here the authors show that in ants such as H. saltator, the 9-exon subfamily of odorant receptors (HsOrs) responds to CHCs, and ectopic expression of HsOrs in Drosophila neurons imparts responsiveness to CHCs.

Biological activity of the enantiomers of 3-methylhentriacontane, a queen pheromone of the ant Lasius niger.

Queen pheromones are essential for regulation of the reproductive division of labor in eusocial insect species. Although only the queen is able to lay fertilized eggs and produce females, in some cases workers may develop their ovaries and lay male-destined eggs, thus reducing the overall colony efficiency. As long as the queen is healthy, it is usually in the workers' collective interest to work for the colony and remain sterile. Queens signal their fertility via pheromones, which may have a primer effect, affecting the physiology of workers, or a releaser effect, influencing worker behavior. The queen pheromone of the ant Lasius niger was among the first queen pheromones of social insects to be identified. Its major component is 3-methylhentriacontane (3-MeC31), which is present in relatively large amounts on the queen's cuticle and on her eggs. 3-MeC31 regulates worker reproduction by inhibiting ovarian development. Most monomethyl-branched hydrocarbons can exist in two stereoisomeric forms. The correct stereochemistry is fundamental to the activity of most bioactive molecules, but this has rarely been investigated for methyl-branched hydrocarbons. Here, we tested the bioactivity of the (S)- and (R)-enantiomers of 3-MeC31, and found that whereas both enantiomers were effective in suppressing worker ovarian development, (S)-3-MeC31 appeared to be more effective at suppressing aggressive behavior by workers. This suggests that the natural pheromone may be a mixture of the two enantiomers. The enantiomeric ratio produced by queens remains unknown because of the small amounts of the compound available from each queen.

Determination of the Absolute Configuration of Female-Produced Contact Sex Pheromone Components of the Longhorned Beetle, Neoclytus acuminatus acuminatus (F).

Cuticular hydrocarbons play important roles in contact chemical communication in insects. Many of these compounds are methyl-branched hydrocarbons with one or more chiral centers, which can exist in two or more stereoisomeric forms. Although the importance of chirality for the volatile semiochemicals that insects use for long-range communication is well established, almost nothing is known about the role of chirality in insect contact chemoreception. Here, we used reverse phase high performance liquid chromatography (RP-HPLC) and digital polarimetry to isolate and determine the absolute configuration of a component of the female-produced contact sex pheromone of the cerambycid beetle, Neoclytus acuminatus acuminatus (F.). The pheromone consists of 7-methylpentacosane (7-MeC25), 7-methylheptacosane (7-MeC27), and 9-methylheptacosane (9-MeC27). The absolute configuration of the most abundant pheromone component, 7-MeC25, was found to be (R). We then utilized enantiomerically pure synthetic pheromone components to test the hypothesis that males would respond more strongly to (R)- than to (S)-enantiomers of the three pheromone components. We also tested blends of (R)-7-MeC27, the most bioactive component, with the (S)-enantiomers of the minor components and vice versa to determine if unnatural stereoisomers might decrease behavioral responses. Males responded most strongly to solvent-washed females treated with the blend of (R)-pheromone components, and to a lesser extent to (R)-7-MeC27 alone. A blend of (R)-7-MeC27 with the (S)-minor components elicited an intermediate response. Together, these findings suggest that the insects can discriminate the absolute configuration of the major and minor pheromone components, and that the configuration of all three components is likely to be (R).

Isolation and determination of absolute configurations of insect-produced methyl-branched hydrocarbons.

Although the effects of stereochemistry have been studied extensively for volatile insect pheromones, little is known about the effects of chirality in the nonvolatile methyl-branched hydrocarbons (MBCHs) used by many insects as contact pheromones. MBCHs generally contain one or more chiral centers and so two or more stereoisomeric forms are possible for each structure. However, it is not known whether insects biosynthesize these molecules in high stereoisomeric purity, nor is it known whether insects can distinguish the different stereoisomeric forms of MBCHs. This knowledge gap is due in part to the lack of methods for isolating individual MBCHs from the complex cuticular hydrocarbon (CHC) blends of insects, as well as the difficulty in determining the absolute configurations of the isolated MBCHs. To address these deficiencies, we report a straightforward method for the isolation of individual cuticular hydrocarbons from the complex CHC blend. The method was used to isolate 36 pure MBCHs from 20 species in nine insect orders. The absolute stereochemistries of the purified MBCHs then were determined by digital polarimetry. The absolute configurations of all of the isolated MBCHs were determined to be (R) by comparison with a library of synthesized, enantiomerically pure standards, suggesting that the biosynthetic pathways used to construct MBCHs are highly conserved within the Insecta. The development of a straightforward method for isolation of specific CHCs will enable determination of their functional roles by providing pure compounds for bioassays.

Elucidating Structure-Bioactivity Relationships of Methyl-Branched Alkanes in the Contact Sex Pheromone of the Parasitic Wasp Lariophagus distinguendus.

The exoskeletons of insects are covered by complex mixtures of cuticular hydrocarbons (CHCs) which are involved in social and sexual communication. However, little is known about the relationship between the structures of CHCs and their behavioral activity. The key component of the contact sex pheromone of the parasitoid Lariophagus distinguendus is 3-methylheptacosane (3-MeC27), which is present in CHC profiles of both females and newly emerged males. The CHCs of females and young males elicit wing-fanning behavior in older males. However, as young males age, 3-MeC27 disappears from their CHC profiles and they no longer elicit wing-fanning responses from other males. We applied enantiopure 3-MeC27 and structurally related CHCs (with respect to chain length or methyl-branch position) to the cuticle of aged male dummies and recorded the wing-fanning behavior of responding males. Only the two enantiomers of 3-MeC27 restored the dummies' attractiveness. The addition of structurally related CHCs or various n-alkanes to bioactive dummies of young males and females significantly decreased wing-fanning by test males. Hence, L. distinguendus males respond specifically but not enantioselectively to 3-MeC27, and perceive the CHC profiles as a whole. Both removal (as is the case with 3-MeC27 in aging males) and addition of individual compounds may disrupt the behavioral response.