Volatile codes: Correlation of olfactory signals and reception in Drosophila-yeast chemical communication.

Drosophila have evolved strong mutualistic associations with yeast communities that best support their growth and survival, resulting in the development of novel niches. It has been suggested that flies recognize their cognate yeasts primarily based on the rich repertoire of volatile organic compounds (VOCs) derived from the yeasts. Thus, it remained an exciting avenue to study whether fly spp. detect and discriminate yeast strains based on odor alone, and if so, how such resolution is achieved by the olfactory system in flies. We used two fly species known to exploit different niches and harboring different yeasts, D. suzukii (a pest of fresh fruit) and D. melanogaster (a saprophytic fly and a neurogenetic model organism). We initially established the behavioral preference of both fly species to six Drosophila-associated yeasts; then chemically analyzed the VOC profile of each yeast which revealed quantitative and qualitative differences; and finally isolated and identified the physiologically active constituents from yeast VOCs for each drosophilid that potentially define attraction. By employing chemical, behavioral, and electrophysiological analyses, we provide a comprehensive portrait of the olfactory neuroethological correlates underlying fly-yeast coadaptation in two drosophilids with distinct habitats

Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants

Odorant-binding proteins (OBPs) were discovered almost three decades ago, but there is still considerable debate regarding their role(s) in insect olfaction, particularly due to our inability to knockdown OBPs and demonstrate their direct phenotypic effects. By using RNA interference (RNAi), we reduced transcription of a major OBP gene, CquiOBP1, in the antennae of the Southern house mosquito, Culex quinquefasciatus. Previously, we had demonstrated that the mosquito oviposition pheromone (MOP) binds to CquiOBP1, which is expressed in MOP-sensitive sensilla. Antennae of RNAi-treated mosquitoes showed significantly lower electrophysiological responses to known mosquito oviposition attractants than the antennae of water-injected, control mosquitoes. While electroantennogram (EAG) responses to MOP, skatole, and indole were reduced in the knockdowns, there was no significant difference in the EAG responses from RNAi-treated and water-injected mosquito antennae to nonanal at all doses tested. These data suggest that CquiOBP1 is involved in the reception of some oviposition attractants, and that high levels of OBPs expression are essential for the sensitivity of the insect’s olfactory system

Molecular Signatures of Sexual Communication in the Phlebotomine Sand Flies

Phlebotomine sand flies employ an elaborate system of pheromone communication wherein males produce pheromones that attract other males to leks (thus acting as an aggregation pheromone) and females to the lekking males (sex pheromone). In addition, the type of pheromone produced varies among populations. Despite the numerous studies on sand fly chemical communication, little is known of their chemosensory genome. Chemoreceptors interact with chemicals in an organism’s environment to elicit essential behaviors such as the identification of suitable mates and food sources. Thus, they play important roles during adaptation and speciation. Major chemoreceptor gene families, odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) together detect and discriminate the chemical landscape. Here, we annotated the chemoreceptor repertoire in the genomes of Lutzomyia longipalpis and Phlebotomus papatasi, major phlebotomine vectors in the New World and Old World, respectively. Comparison with other sequenced Diptera revealed a large and unique expansion where over 80% of the ~140 ORs belong to a single, taxonomically restricted clade. We next conducted a comprehensive analysis of the chemoreceptors in 63 L. longipalpis individuals from four different locations in Brazil representing allopatric and sympatric populations and three sex-aggregation pheromone types (chemotypes). Population structure based on single nucleotide polymorphisms (SNPs) and gene copy number in the chemoreceptors corresponded with their putative chemotypes, and corroborate previous studies that identified multiple populations. Our work provides genomic insights into the underlying behavioral evolution of sexual communication in the L. longipalpis species complex in Brazil, and highlights the importance of accounting for the ongoing speciation in central and South American Lutzomyia that could have important implications for vectorial capacity

Pheromone Binding to General Odorant-binding Proteins from the Navel Orangeworm

General odorant-binding proteins (GOBPs) of moths are postulated to be involved in the reception of semiochemicals other than sex pheromones, the so-called “general odorants.” We have expressed two GOBPs, AtraGOBP1 and AtraGOBP2, which were previously isolated from the antennae of the navel orangeworm, Amyelois transitella. Surprisingly, these two proteins did not bind compounds that are known to attract adult moths, particularly females. The proper folding and functionality of the recombinant proteins was inferred from circular dichroism analysis and demonstration that both GOBPs bound nonanal in a pH-dependent manner. EAG experiments demonstrated that female attractants (1-phenylethanol, propionic acid phenyl ester, and isobutyric acid phenyl ester) are detected with high sensitivity by the antennae of day-0 to day-4 adult females, with response declining in older moths. The same age-dependence was shown for male antennae responding to constituents of the sex pheromone. Interestingly, AtraGOBP2 bound the major constituent of the sex pheromone, Z11Z13-16Ald, with affinity comparable to that shown by a pheromone-binding protein, AtraPBP1. The related alcohol bound to AtraPBP1 with higher affinity than to AtraGOBP2. AtraGOBP1 bound both ligands with low but nearly the same affinity

Generic Insect Repellent Detector from the Fruit Fly Drosophila melanogaster

Background: Insect repellents are prophylactic tools against a number of vector-borne diseases. There is growing demand for repellents outperforming DEET in cost and safety, but with the current technologies R&D of a new product takes almost 10 years, with a prohibitive cost of $30 million dollar in part due to the demand for large-scale synthesis of thousands of test compounds of which only 1 may reach the market. R&D could be expedited and cost dramatically reduced with a molecular/physiological target to streamline putative repellents for final efficacy and toxicological tests. Methodology: Using olfactory-based choice assay we show here that the fruit fly is repelled by not only DEET, but also IR3535 and picaridin thus suggesting they might have ‘‘generic repellent detector(s),’ ’ which may be of practical applications in new repellent screenings. We performed single unit recordings from all olfactory sensilla in the antennae and maxillary palps. Although the ab3A neuron in the wild type flies responded to picaridin, it was unresponsive to DEET and IR3535. By contrast, a neuron housed in the palp basiconic sensilla pb1 responded to DEET, IR3535, and picaridin, with apparent sensitivity higher than that of the DEET detectors in the mosquitoes Culex quinquefasciatus and Aedes aegypti. DmOr42a was transplanted from pb1 to the ‘‘empty neuron’ ’ and showed to be sensitive to the three insect repellents. Conclusions: For the first time we have demonstrated that the fruit fly avoids not only DEET but also IR3535 and picaridin, and identified an olfactory receptor neuron (ORN), which is sensitive to these three major insect repellents. We have als

Reverse and Conventional Chemical Ecology Approaches for the Development of Oviposition Attractants for Culex Mosquitoes

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 µg/l) and nonanal (0.15 ng/µl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture

Specificity of the receptor for the major sex pheromone component in Heliothis virescens.

In a previous study, the Drosophila melanogaster OR67d(GAL4);UAS system was used to functionally characterize the receptor for the major component of the sex pheromone in the tobacco budworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae), HvOR13. Electrophysiological and behavioral assays showed that transgenic flies expressing HvOR13 responded to (Z)-11-hexadecenal (Z11-16:Ald). However, tests were not performed to determine whether these flies would also respond to secondary components of the H. virescens sex pheromone. Thus, in this study the response spectrum of HvOR13 expressed in this system was examined by performing single cell recordings from odor receptor neuron in trichoid T1 sensilla on antennae of two Or67d(GAL4 [1]); UAS-HvOR13 lines stimulated with Z11-16:Ald and six H. virescens secondary pheromone components. Fly courtship assays were also performed to examine the behavioral response of the Or67d(GAL4[1]); UAS-HvOR13 flies to Z11-16:Ald and the secondary component Z9-14:Ald. Our combined electrophysiological and behavioral studies indicated high specificity and sensitivity of HvOR13 to Z11-16:Ald. Interestingly, a mutation leading to truncation in the HvOR13 C-terminal region affected but did not abolish pheromone receptor response to Z11-16:Ald. The findings are assessed in relationship to other HvOR13 heterologous expression studies, and the role of the C-terminal domain in receptor function is discussed. A third line expressing HvOR15 was also tested but did not respond to any of the seven pheromone components

Chemical Ecology of Oviposition Dynamics in Drosophila suzukii (Diptera: Drosophilidae)

Massive economic damage by spotted-wing drosophila (SWD), Drosophila suzukii, results from their unique egg laying behavior wherein a gravid fly pierces a ripening fruit to lay a number of eggs. Gravid SWD flies employ a complex suite of behaviors to find a fruit with the optimum firmness and chemistry. We investigated chemosensory cues potentially contributing to the oviposition behavior of SWD. In a series of experiments, we studied how the fruit ripeness and the underlying volatile chemistry influence oviposition. We tested the comparative attraction of three common fruits implicated in oviposition and determined raspberries to be most attractive in the trap choice assays that strictly measured olfactory preference. Since SWD oviposit in ripening fruits and appear to avoid the overripe fruit, we further evaluated the effect of ripeness on gravid fly attraction. Overripe fruits were significantly more attractive compared to the ripe fruits. The trap choice assays were repeated in an olfactory T-maze paradigm that provided a complex odor environment, potentially experienced by the gravid flies, and the results were mostly comparable. Since our behavioral paradigms indicated a clear olfactory preference for specific ripeness stages (ripe and overripe), we analyzed the constituent volatile odorants from the three ripening stages, revealing discrete odor profiles. Finally, we quantified the total soluble sugars and carbon dioxide concentrations from field-collected raspberries in underripe, ripe, and overripe conditions, revealing that the overripe stage is the most sugar-rich. Together, our results indicate unique chemosensory adaptations in gravid SWD flies for successfully exploiting optimal oviposition resources