BackgroundChemosensory signal transduction guides the behavior of many insects, including Anopheles gambiae, the major vector for human malaria in sub-Saharan Africa. To better understand the molecular basis of mosquito chemosensation we have used whole transcriptome RNA sequencing (RNA-seq) to compare transcript expression profiles between the two major chemosensory tissues, the antennae and maxillary palps, of adult female and male An. gambiae.ResultsWe compared chemosensory tissue transcriptomes to whole body transcriptomes of each sex to identify chemosensory enhanced genes. In the six data sets analyzed, we detected expression of nearly all known chemosensory genes and found them to be highly enriched in both olfactory tissues of males and females. While the maxillary palps of both sexes demonstrated strict chemosensory gene expression overlap, we observed acute differences in sensory specialization between male and female antennae. The relatively high expression levels of chemosensory genes in the female antennae reveal its role as an organ predominately assigned to chemosensation. Remarkably, the expression of these genes was highly conserved in the male antennae, but at much lower relative levels. Alternatively, consistent with a role in mating, the male antennae displayed significant enhancement of genes involved in audition, while the female enhancement of these genes was observed, but to a lesser degree.ConclusionsThese findings suggest that the chemoreceptive spectrum, as defined by gene expression profiles, is largely similar in female and male An. gambiae. However, assuming sensory receptor expression levels are correlated with sensitivity in each case, we posit that male and female antennae are perceptive to the same stimuli, but possess inverse receptive prioritizations and sensitivities. Here we have demonstrated the use of RNA-seq to characterize the sensory specializations of an important disease vector and grounded future studies investigating chemosensory processes.

In insects, odor cues are discriminated through a divergent family of odorant receptors (ORs). A functional OR complex consists of both a conventional odorant-binding OR and a nonconventional coreceptor (Orco) that is highly conserved across insect taxa. Recent reports have characterized insect ORs as ion channels, but the precise mechanism of signaling remains unclear. We report the identification and characterization of an Orco family agonist, VUAA1, using the Anopheles gambiae coreceptor (AgOrco) and other orthologues. These studies reveal that the Orco family can form functional ion channels in the absence of an odor-binding OR, and in addition, demonstrate a first-in-class agonist to further research in insect OR signaling. In light of the extraordinary conservation and widespread expression of the Orco family, VUAA1 represents a powerful new family of compounds that can be used to disrupt the destructive behaviors of nuisance insects, agricultural pests, and disease vectors alike.

A combination of gene silencing and behavioral studies in the malaria vector mosquito Anopheles gambiae sheds light on the olfactory basis of DEET repulsion as well as reveals the role of another family of chemosensory receptors that facilitate olfaction in An. gambiae.

Threshold photodetachment cross sections for OH− and OD− at photon energies close to their electron binding energies have been obtained using a crossed tunable laser-negative ion beam apparatus at a resolution of 25 μeV. The data reveal many clear features associated with photodetachment of single rotational levels of the negative ion, producing single rotational levels of the neutral and a near zero energy electron. From the frequencies of the observed thresholds, the spectroscopic constants of OH− and OD− are found. The rotational constants of the vibrational ground state are B0 = 18.7409(45) cm−1 and D0 = 2.052(45)×10−3 cm−1 for OH− and B0 = 9.9852(48) cm−1 and D0 = 0.553(33)×10−3 cm−1 for OD−. The electron affinities of OH and OD are measured to be 14 741.03(17) cm−1 and 14 723.92(30) cm−1, respectively. The observed bound–free transitions satisfy selection rules only slightly different from the selection rules observed in bound–bound spectroscopy. The relative intensities of the transitions show the transition from Hund’s case (a) to case (b) as the rotation in OH increases. The intensities are in agreement with a theory based on an intermediate complex which dissociates into jj-coupled constituents. The shape of the cross section near threshold, a probe of the long range electron–OH interaction, is found to depend on the final rotational state of the OH neutral. This dependence is studied in detail. Analogous studies on photodetachment of OD− confirm our deductions.

Aedes aegypti and Anopheles gambiae are among the best-characterized mosquito species within the Culicinae and Anophelinae mosquito clades which diverged ∼150 million years ago. Despite this evolutionary distance, the olfactory systems of these mosquitoes exhibit similar morphological and physiological adaptations. Paradoxically, mosquito odorant receptors, which lie at the heart of chemosensory signal transduction pathways, belong to a large and highly divergent gene family. We have used 2 heterologous expression systems to investigate the functional characteristics of a highly conserved subset of Ors between Ae. aegypti and An. gambiae to investigate whether protein homology correlates with odorant-induced activation. We find that these receptors share similar odorant response profiles and that indole, a common and ecologically relevant olfactory cue, elicits strong responses from these homologous receptors. The identification of other highly conserved members of this Or clade from mosquito species of varying phylogenetic relatedness supports a model in which high sensitivity to indole represents an ancient ecological adaptation that has been preserved as a result of its life cycle importance. These results provide an understanding of how similarities and disparities among homologous OR proteins relate to olfactory function, which can lead to greater insights into the design of successful strategies for the control of mosquito-borne diseases.

BackgroundAt a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1.ResultsA search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation.ConclusionsWe have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.

BackgroundInsect odorant receptors (ORs) function as odorant-gated ion channels consisting of a conventional, odorant-binding OR and the Orco coreceptor. While Orco can function as a homomeric ion channel, the role(s) of the conventional OR in heteromeric OR complexes has largely focused only on odorant recognition.ResultsTo investigate other roles of odorant-binding ORs, we have employed patch clamp electrophysiology to investigate the properties of the channel pore of several OR complexes formed by a range of different odorant-specific Anopheles gambiae ORs (AgOrs) each paired with AgOrco. These studies reveal significant differences in cation permeability and ruthenium red susceptibility among different AgOr complexes.ConclusionsWith observable differences in channel function, the data support a model in which the odorant-binding OR also affects the channel pore. The variable effect contributed by the conventional OR on the conductive properties of odorant-gated sensory channels adds additional complexity to insect olfactory signaling, with differences in odor coding beginning with ORs on the periphery of the olfactory system.

Agonism of insect odorant receptor (OR) cation channels may represent a new strategy for the manipulation of destructive insect olfactory-driven behaviors. We have explored the chemical space around VUAA1, the first in class agonist of the obligate OR co-receptor ion channel (Orco), and describe novel compound analogues with increased potency across insect taxa. Functional analyses reveal several of these VUAA1 structural analogues display significantly greater potency as compared to the activity of the previously described active compounds in mobility-based behavioral assays on mosquito larvae.