David C. Rinker

Functional agonism of insect odorant receptor ion channels

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.

Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue- and sex-specific signatures of odor coding

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.

Blood meal-induced changes to antennal transcriptome profiles reveal shifts in odor sensitivities in Anopheles gambiae

Olfactory-driven behaviors are central to the lifecycle of the malaria vector mosquito Anopheles gambiae and are initiated by peripheral signaling in the antenna and other olfactory tissues. To continue gaining insight into the relationship between gene expression and olfaction, we have performed cohort comparisons of antennal transcript abundances at five time points after a blood meal, a key event in both reproduction and disease transmission cycles. We found that more than 5,000 transcripts displayed significant abundance differences, many of which were correlated by cluster analysis. Within the chemosensory gene families, we observed a general reduction in the level of chemosensory gene transcripts, although a subset of odorant receptors (AgOrs) was modestly enhanced in post–blood-fed samples. Integration of AgOr transcript abundance data with previously characterized AgOr excitatory odorant response profiles revealed potential changes in antennal odorant receptivity that coincided with the shift from host-seeking to oviposition behaviors in blood-fed female mosquitoes. Behavioral testing of ovipositing females to odorants highlighted by this synthetic analysis identified two unique, unitary oviposition cues for An. gambiae, 2-propylphenol and 4-methylcyclohexanol. We posit that modest, yet cumulative, alterations of AgOr transcript levels modulate peripheral odor coding resulting in biologically relevant behavioral effects. Moreover, these results demonstrate that highly quantitative, RNAseq transcript abundance data can be successfully integrated with functional data to generate testable hypotheses.

Heteromeric Anopheline Odorant Receptors Exhibit Distinct Channel Properties

Background Insect 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. Results To 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. Conclusions With 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.

Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae

BackgroundTwo sibling members of the Anopheles gambiae species complex display notable differences in female blood meal preferences. An. gambiae s.s. has a well-documented preference for feeding upon human hosts, whereas An. quadriannulatus feeds on vertebrate/mammalian hosts, with only opportunistic feeding upon humans. Because mosquito host-seeking behaviors are largely driven by the sensory modality of olfaction, we hypothesized that hallmarks of these divergent host seeking phenotypes will be in evidence within the transcriptome profiles of the antennae, the mosquito’s principal chemosensory appendage.ResultsTo test this hypothesis, we have sequenced antennal mRNA of non-bloodfed females from each species and observed a number of distinct quantitative and qualitative differences in their chemosensory gene repertoires. In both species, these gene families show higher rates of sequence polymorphisms than the overall rates in their respective transcriptomes, with potentially important divergences between the two species. Moreover, quantitative differences in odorant receptor transcript abundances have been used to model potential distinctions in volatile odor receptivity between the two sibling species of anophelines.ConclusionThis analysis suggests that the anthropophagic behavior of An. gambiae s.s. reflects the differential distribution of olfactory receptors in the antenna, likely resulting from a co-option and refinement of molecular components common to both species. This study improves our understanding of the molecular evolution of chemoreceptors in closely related anophelines and suggests possible mechanisms that underlie the behavioral distinctions in host seeking that, in part, account for the differential vectorial capacity of these mosquitoes.

The genomics of microbial domestication in the fermented food environment

Shortly after the agricultural revolution, the domestication of bacteria, yeasts, and molds, played an essential role in enhancing the stability, quality, flavor, and texture of food products. These domestication events were probably the result of human food production practices that entailed the continual recycling of isolated microbial communities in the presence of abundant agricultural food sources. We suggest that within these novel agrarian food niches the metabolic requirements of those microbes became regular and predictable resulting in rapid genomic specialization through such mechanisms as pseudogenization, genome decay, interspecific hybridization, gene duplication, and horizontal gene transfer. The ultimate result was domesticated strains of microorganisms with enhanced fermentative capacities.

Novel high-throughput screens of Anopheles gambiae odorant receptors reveal candidate behaviour-modifying chemicals for mosquitoes

Despite many decades of multilateral global efforts, a significant portion of the world population continues to be plagued with one or more mosquito‐vectored diseases. These include malaria and filariasis, as well as numerous arboviral‐associated illnesses, such as dengue and yellow fevers. The dynamics of disease transmission by mosquitoes is complex, and involves both vector competence and vectorial capacity. One area of intensive effort is the study of chemosensory‐driven behaviours in the malaria vector mosquito Anopheles gambiae Giles, the modulation of which is likely to provide opportunities for disease reduction. In this context, recent studies characterize a large divergent family of An. gambiae odorant receptors (AgORs) that play critical roles in olfactory signal transduction. This work facilitates high‐throughput, cell‐based calcium mobilization screens of AgOR‐expressing human embryonic kidney cells identifying a large number of conventional AgOR ligands, as well as the first nonconventional Orco (olfactory receptor co‐receptor) family agonist. As such, ligand‐mediated modulation serves as a proof‐of‐concept demonstration that AgORs represent viable targets for high‐throughput screening and for the eventual development of behaviour‐modifying olfactory compounds. Such attractants or repellents could foster malaria reduction programmes.

Divergent and Conserved Elements Comprise the Chemoreceptive Repertoire of the Nonblood-Feeding Mosquito Toxorhynchites amboinensis

Many mosquito species serve as vectors of diseases such as malaria and yellow fever, wherein pathogen transmission is tightly associated with the reproductive requirement of taking vertebrate blood meals. Toxorhynchites is one of only three known mosquito genera that does not host-seek and initiates egg development in the absence of a blood-derived protein bolus. These remarkable differences make Toxorhynchites an attractive comparative reference for understanding mosquito chemosensation as it pertains to host-seeking. We performed deep transcriptome profiling of adult female Toxorhynchites amboinensis bodies, antennae and maxillary palps, and identified 25,084 protein-coding “genes” in the de novo assembly. Phylogenomic analysis of 4,266 single-copy “genes” from T. amboinensis, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus robustly supported Ae. aegypti as the closest relative of T. amboinensis, with the two species diverged approximately 40 Ma. We identified a large number of T. amboinensis chemosensory “genes,” the majority of which have orthologs in other mosquitoes. Finally, cross-species expression analyses indicated that patterns of chemoreceptor transcript abundance were very similar for chemoreceptors that are conserved between T. amboinensis and Ae. aegypti, whereas T. amboinensis appeared deficient in the variety of expressed, lineage-specific chemoreceptors. Our transcriptome assembly of T. amboinensis represents the first comprehensive genomic resource for a nonblood-feeding mosquito and establishes a foundation for future comparative studies of blood-feeding and nonblood-feeding mosquitoes. We hypothesize that chemosensory genes that display discrete patterns of evolution and abundance between T. amboinensis and blood-feeding mosquitoes are likely to play critical roles in host-seeking and hence the vectorial capacity.

Transcriptional profile and differential fitness in a specialist milkweed insect across host plants varying in toxicity

Interactions between plants and herbivorous insects have been models for theories of specialization and co‐evolution for over a century. Phytochemicals govern many aspects of these interactions and have fostered the evolution of adaptations by insects to tolerate or even specialize on plant defensive chemistry. While genomic approaches are providing new insights into the genes and mechanisms insect specialists employ to tolerate plant secondary metabolites, open questions remain about the evolution and conservation of insect counterdefences, how insects respond to the diversity defences mounted by their host plants, and the costs and benefits of resistance and tolerance to plant defences in natural ecological communities. Using a milkweed‐specialist aphid (Aphis nerii) model, we test the effects of host plant species with increased toxicity, likely driven primarily by increased secondary metabolites, on aphid life history traits and whole‐body gene expression. We show that more toxic plant species have a negative effect on aphid development and lifetime fecundity. When feeding on more toxic host plants with higher levels of secondary metabolites, aphids regulate a narrow, targeted set of genes, including those involved in canonical detoxification processes (e.g., cytochrome P450s, hydrolases, UDP‐glucuronosyltransferases and ABC transporters). These results indicate that A. nerii marshal a variety of metabolic detoxification mechanisms to circumvent milkweed toxicity and facilitate host plant specialization, yet, despite these detoxification mechanisms, aphids experience reduced fitness when feeding on more toxic host plants. Disentangling how specialist insects respond to challenging host plants is a pivotal step in understanding the evolution of specialized diet breadths.

RNAseq in the mosquito maxillary palp: a little antennal RNA goes a long way

A comparative transcriptomic study of mosquito olfactory tissues recently published in BMC Genomics (Hodges et al, 2014) reported several novel findings that have broad implications for the field of insect olfaction. In this brief commentary, we outline why the conclusions of Hodges et al. are problematic under the current models of insect olfaction and then contrast their findings with those of other RNAseq based studies of mosquito olfactory tissues. We also generated a new RNAseq data set from the maxillary palp of Anopheles gambiae in an effort to replicate the novel results of Hodges et al. but were unable to reproduce their results. Instead, our new RNAseq data support the more straightforward explanation that the novel findings of Hodges et al. were a consequence of contamination by antennal RNA. In summary, we find strong evidence to suggest that the conclusions of Hodges et al were spurious, and that at least some of their RNAseq data sets were irrevocably compromised by cross-contamination between samples.