Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila.

Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasitoid wasp species kill a vast range of insect species. Insects have evolved defensive responses to the threat of wasps, some cellular and some behavioral. Here we find an unexpected response of adult Drosophila to the presence of certain parasitoid wasps: accelerated mating behavior. Flies exposed to certain wasp species begin mating more quickly. The effect is mediated via changes in the behavior of the female fly and depends on visual perception. The sight of wasps induces the dramatic upregulation in the fly nervous system of a gene that encodes a 41-amino acid micropeptide. Mutational analysis reveals that the gene is essential to the behavioral response of the fly. Our work provides a foundation for further exploration of how the activation of visual circuits by the sight of a wasp alters both sexual behavior and gene expression.

4-Vinylanisole is an aggregation pheromone in locusts.

Locust plagues threaten agricultural and environmental safety throughout the world1,2. Aggregation pheromones have a crucial role in the transition of locusts from a solitary form to the devastating gregarious form and the formation of large-scale swarms3,4. However, none of the candidate compounds reported5-7 meet all the criteria for a locust aggregation pheromone. Here, using behavioural assays, electrophysiological recording, olfactory receptor characterization and field experiments, we demonstrate that 4-vinylanisole (4VA) (also known as 4-methoxystyrene) is an aggregation pheromone of the migratory locust (Locusta migratoria). Both gregarious and solitary locusts are strongly attracted to 4VA, regardless of age and sex. Although it is emitted specifically by gregarious locusts, 4VA production can be triggered by aggregation of four to five solitary locusts. It elicits responses specifically from basiconic sensilla on locust antennae. We also identified OR35 as a specific olfactory receptor of 4VA. Knockout of OR35 using CRISPR-Cas9 markedly reduced the electrophysiological responses of the antennae and impaired 4VA behavioural attractiveness. Finally, field trapping experiments verified the attractiveness of 4VA to experimental and wild populations. These findings identify a locust aggregation pheromone and provide insights for the development of novel control strategies for locusts.

Robust olfactory responses in the absence of odorant binding proteins.

Odorant binding proteins (Obps) are expressed at extremely high levels in the antennae of insects, and have long been believed essential for carrying hydrophobic odorants to odor receptors. Previously we found that when one functional type of olfactory sensillum in Drosophila was depleted of its sole abundant Obp, it retained a robust olfactory response (Larter et al., 2016). Here we have deleted all the Obp genes that are abundantly expressed in the antennal basiconic sensilla. All of six tested sensillum types responded robustly to odors of widely diverse chemical or temporal structure. One mutant gave a greater physiological and behavioral response to an odorant that affects oviposition. Our results support a model in which many sensilla can respond to odorants in the absence of Obps, and many Obps are not essential for olfactory response, but that some Obps can modulate olfactory physiology and the behavior that it drives.

OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.

Asprosin is a fasting-induced hormone that promotes glucose production in the liver and stimulates appetite in the hypothalamus by activating the cAMP signaling pathway via an unknown G protein-coupled receptor (GPCR). However, the bona fide receptor of Asprosin is unclear. Here, we have identified that the olfactory receptor OLFR734 acts as a receptor of Asprosin to modulate hepatic glucose production. Olfr734 knockout mice show a blunted response to Asprosin, including attenuated cAMP levels and hepatic glucose production, and improved insulin sensitivity. As Olfr734 deficiency dramatically attenuates both fasting and high-fat-diet-induced glucose production, our results demonstrate a critical role of OLFR734 as a receptor of Asprosin to maintain glucose homeostasis during fasting and in obesity.

Molecular and functional characterization of odorant-binding protein genes in Holotrichia oblita Faldermann.

Holotrichia oblita is one of the nastiest pests in China. In present research, four full-length cDNA encoding of HoblOBP genes were cloned and sequenced from H. oblita. The mRNA of HoblOBPs were predominantly expressed in antenna. The recombinant HoblOBPs proteins were obtained for fluorescence binding assays. Four of HoblOBPs could mediate the response of H. oblita to organic fertilizers-derived attractants, including HoblOBP5 binding to skatole; HoblOBP8 binding to p-cresol, indole and skatole; HoblOBP9 binding to indole and 4-allylanisole; and HoblOBP24 binding to p-cresol, indole and 4-ethylphenol. Further, RNA interference demonstrated that transcripts of HoblOBP5, 8, 9, and 24 decreased in a time-dependent manner after dsRNA-injection. Knockdown of HoblOBP5, 8, 9, and 24 by injection of dsRNA successfully interfered with behavioral responses towards the target compounds in beetles. Our results showed that HoblOBP5, HoblOBP8, HoblOBP9 and HoblOBP24 are essential in mediating the approach behavior of H. oblita.

Oxygen regulation of breathing through an olfactory receptor activated by lactate.

Animals have evolved homeostatic responses to changes in oxygen availability that act on different timescales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long-term responses to low oxygen (hypoxia) has been established, the pathway that mediates acute responses to hypoxia in mammals is not well understood. Here we show that the olfactory receptor gene Olfr78 is highly and selectively expressed in oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation that monitors blood oxygen and stimulates breathing within seconds when oxygen declines. Olfr78 mutants fail to increase ventilation in hypoxia but respond normally to hypercapnia. Glomus cells are present in normal numbers and appear structurally intact, but hypoxia-induced carotid body activity is diminished. Lactate, a metabolite that rapidly accumulates in hypoxia and induces hyperventilation, activates Olfr78 in heterologous expression experiments, induces calcium transients in glomus cells, and stimulates carotid sinus nerve activity through Olfr78. We propose that, in addition to its role in olfaction, Olfr78 acts as a hypoxia sensor in the breathing circuit by sensing lactate produced when oxygen levels decline.

Non-redundant coding of aversive odours in the main olfactory pathway.

Many species are critically dependent on olfaction for survival. In the main olfactory system of mammals, odours are detected by sensory neurons that express a large repertoire of canonical odorant receptors and a much smaller repertoire of trace amine-associated receptors (TAARs). Odours are encoded in a combinatorial fashion across glomeruli in the main olfactory bulb, with each glomerulus corresponding to a specific receptor. The degree to which individual receptor genes contribute to odour perception is unclear. Here we show that genetic deletion of the olfactory Taar gene family, or even a single Taar gene (Taar4), eliminates the aversion that mice display to low concentrations of volatile amines and to the odour of predator urine. Our findings identify a role for the TAARs in olfaction, namely, in the high-sensitivity detection of innately aversive odours. In addition, our data reveal that aversive amines are represented in a non-redundant fashion, and that individual main olfactory receptor genes can contribute substantially to odour perception.

Characterization of the 5' and 3' breakpoints of the Spanish (뫧)0-thalassemia deletion in Mexican patients.

We studied five unrelated Mexican carriers of the Spanish (δß)(0)-thalassemia [(δß)(0)-thal] mutation to characterize the size of the deletion, the 5' and 3' breakpoints and the 5' ß-globin haplotype. Sequence analysis revealed the presence of an 89,548 bp deletion. The δ- and ß-globin genes, two olfactory receptor genes (OR51V1 and OR52A1) and two pseudogenes (OR52Z1P and OR51A1P) were deleted. The 5' breakpoint was located at the same position as previously reported, and the 3' breakpoint was situated 7.0 kb downstream of OR52A1 and 11.7 kb upstream of OR52A5. The Spanish (δß)(0)-thal allele was associated with the 5' haplotype 2 [- + + - +] in the studied patients. Because this mutation is relatively frequent in Spain, and the Mexican population contains a high level of Spanish genetic background, we propose that the mutation in both populations share a common ancestral origin.

Adaptive adjustment of the generalization-discrimination balance in larval Drosophila.

Learnt predictive behavior faces a dilemma: predictive stimuli will never 'replay' exactly as during the learning event, requiring generalization. In turn, minute differences can become meaningful, prompting discrimination. To provide a study case for an adaptive adjustment of this generalization-discrimination balance, the authors ask whether Drosophila melanogaster larvae are able to either generalize or discriminate between two odors (1-octen-3-ol and 3-octanol), depending on the task. The authors find that after discriminatively rewarding one but not the other odor, larvae show conditioned preference for the rewarded odor. On the other hand, no odor specificity is observed after nondiscriminative training, even if the test involves a choice between both odors. Thus, for this odor pair at least, discrimination training is required to confer an odor-specific memory trace. This requires that there is at least some difference in processing between the two odors already at the beginning of the training. Therefore, as a default, there is a small yet salient difference in processing between 1-octen-3-ol and 3-octanol; this difference is ignored after nondiscriminative training (generalization), whereas it is accentuated by odor-specific reinforcement (discrimination). Given that, as the authors show, both faculties are lost in anosmic Or83b(1) mutants, this indicates an adaptive adjustment of the generalization-discrimination balance in larval Drosophila, taking place downstream of Or83b-expressing sensory neurons.

State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging.

Gene-targeted deletion of the predominant Shaker potassium channel, Kv1.3, in the mitral cells of the olfactory bulb, decreases the number of presynaptic, odorant receptor (OR)-identified olfactory sensory neurons (OSNs) in the main olfactory epithelium (MOE) and alters the nature of their postsynaptic connections to mitral cell targets. The current study examined whether OSN density was state-dependent by examining the impact of (1) odor enrichment, (2) sensory deprivation, and (3) aging upon the number of P2- or M72-expressing neurons. Histological approaches were used to quantify the number of OSNs across entire epithelia for wildtype (WT) vs. Kv1.3-null (KO) mice bred onto an ORtauLacZ reporter background. Following either odor enrichment or early unilateral naris-occlusion, the number of M72-expressing OSNs was significantly decreased in WT mice, but was unchanged in KO animals. Following naris-occlusion, the number of P2-expressing OSNs was decreased regardless of genotype. Animals that were reared to 2 years of age demonstrated loss of both P2- and M72-expressing OSNs in WT mice and a concomitant loss of only M72-expressing neurons in KO mice. These findings suggest that voltage-gated activity of the mitral cells is important for OSN plasticity, and can prevent neuronal loss via sensory- and OR-dependent mechanisms.

The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor.

Various social behaviours in mice are regulated by chemical signals called pheromones that act through the vomeronasal system. Exocrine gland-secreting peptide 1 (ESP1) is a 7-kDa peptide that is released into male tear fluids and stimulates vomeronasal sensory neurons in female mice. Here, we describe the molecular and neural mechanisms that are involved in the decoding of ESP1 signals in the vomeronasal system, which leads to behavioural output in female mice. ESP1 is recognized by a specific vomeronasal receptor, V2Rp5, and the ligand-receptor interaction results in sex-specific signal transmission to the amygdaloid and hypothalamic nuclei via the accessory olfactory bulb. Consequently, ESP1 enhances female sexual receptive behaviour upon male mounting (lordosis), allowing successful copulation. In V2Rp5-deficient mice, ESP1 induces neither neural activation nor sexual behaviour. These findings show that ESP1 is a crucial male pheromone that regulates female reproductive behaviour through a specific receptor in the mouse vomeronasal system.

Regulation of odor receptor genes in trichoid sensilla of the Drosophila antenna.

This study concerns the problem of odor receptor gene choice in the fruit fly Drosophila melanogaster. From a family of 60 Odor receptor genes, only one or a small number are selected for expression by each olfactory receptor neuron. Little is known about how an olfactory receptor neuron selects a receptor, or how the nucleotide sequences flanking a receptor gene dictate its expression in a particular neuron. Previous investigation has primarily concerned the maxillary palp, the simpler of the fly's two olfactory organs. Here we focus on genes encoding four antennal receptors that respond to fly odors in an in vivo expression system. To investigate the logic of odor receptor expression, we carry out a genetic analysis of their upstream regulatory sequences. Deletion analysis reveals that relatively short regulatory regions are sufficient to confer expression in the appropriate neurons, with limited if any misexpression. We find evidence for both positive and negative regulation. Multiple repressive functions restrict expression to the antenna, to a region of the antenna, and to neurons. Through deletion and base substitution mutagenesis we identify GCAATTA elements and find evidence that they act in both positive and negative regulation.

Distinct functions of acj6 splice forms in odor receptor gene choice.

Individual olfactory receptor neurons (ORNs) selectively express one or a small number of odor receptors from among a large receptor repertoire. The expression of an odor receptor dictates the odor response spectrum of the ORN. The process of receptor gene choice relies in part on a combinatorial code of transcription factors. In Drosophila, the POU domain transcription factor Acj6 is one element of the transcription factor code. In acj6 null mutants, many ORNs do not express an appropriate odor receptor gene and thus are not correctly specified. We find that acj6 is alternatively spliced to yield many structurally distinct transcripts in the olfactory organs. We generate flies that express single splice forms of acj6 in an acj6(-) background. We find that different splice forms are functionally distinct; they differ in their abilities to specify ORN identities. Some individual splice forms can fully rescue the specification of some ORNs. Individual splice forms can function both positively and negatively in receptor gene regulation. ORNs differ in their requirements for splice forms; some are not fully rescued by any single splice form tested, suggesting that some ORNs may require the combinatorial action of multiple splice forms. Late expression of some acj6 splice forms is sufficient to rescue some ORN classes, consistent with a direct role for Acj6 isoforms in receptor gene expression. The results indicate that alternative splicing may add another level of richness to the regulatory code that underlies the process of odor receptor gene choice.

Neuronal activity and Wnt signaling act through Gsk3-beta to regulate axonal integrity in mature Drosophila olfactory sensory neurons.

The roles played by signaling pathways and neural activity during the development of circuits have been studied in several different contexts. However, the mechanisms involved in maintaining neuronal integrity once circuits are established are less well understood, despite their potential relevance to neurodegeneration. We demonstrate that maintenance of adult Drosophila olfactory sensory neurons requires cell-autonomous neuronal activity. When activity is silenced, development occurs normally, but neurons degenerate in adulthood. These detrimental effects can be compensated by downregulating Glycogen synthase kinase-3beta (Gsk-3beta). Conversely, ectopic expression of activated Gsk-3beta or downregulation of Wnt effectors also affect neuron stability, demonstrating a role for Wnt signaling in neuroprotection. This is supported by our observation that activated adult neurons are capable of increased Wingless release, and its targeted expression can protect neurons against degeneration. The role of Wnt signaling in this process is non-transcriptional, and may act on cellular mechanisms that regulate axonal or synaptic stability. Together, we provide evidence that Gsk-3beta is a key sensor involved in neural circuit integrity, maintaining axon stability through neural activity and the Wnt pathway.

Translation of sensory input into behavioral output via an olfactory system.

We investigate the logic by which sensory input is translated into behavioral output. First we provide a functional analysis of the entire odor receptor repertoire of an olfactory system. We construct tuning curves for the 21 functional odor receptors of the Drosophila larva and show that they sharpen at lower odor doses. We construct a 21-dimensional odor space from the responses of the receptors and find that the distance between two odors correlates with the extent to which one odor masks the other. Mutational analysis shows that different receptors mediate the responses to different concentrations of an odorant. The summed response of the entire receptor repertoire correlates with the strength of the behavioral response. The activity of a small number of receptors is a surprisingly powerful predictor of behavior. Odors that inhibit more receptors are more likely to be repellents. Odor space is largely conserved between two dissimilar olfactory systems.

Deletion of the core-H region in mice abolishes the expression of three proximal odorant receptor genes in cis.

We have previously reported that a 2.1-kb homology (H) sequence, conserved between mouse and human, regulates the odorant receptor (OR) gene MOR28 in transgenic mice. Here, we narrowed down the essential sequences of the H to a core of 124 bp by using a transient expression system in zebrafish embryos. Transgenic experiments in mice demonstrated that the core-H sequence is sufficient to endow expression of the MOR28 minigene. Deletion and mutation analyses of the core-H region revealed two homeodomain sequences to be essential for the H enhancer activity. Targeted deletion of the core-H abolished expression of three proximal OR genes, MOR28, MOR10, and MOR83, in cis, indicating the presence of another locus control region/enhancer in the downstream region, that regulates four distal OR genes in the same MOR28 cluster. In the heterozygous mice, the H(-) phenotype of the mutant allele was not rescued by the wild-type H(+) allele in trans.

The molecular basis of odor coding in the Drosophila antenna.

We have undertaken a functional analysis of the odorant receptor repertoire in the Drosophila antenna. Each receptor was expressed in a mutant olfactory receptor neuron (ORN) used as a "decoder," and the odor response spectrum conferred by the receptor was determined in vivo by electrophysiological recordings. The spectra of these receptors were then matched to those of defined ORNs to establish a receptor-to-neuron map. In addition to the odor response spectrum, the receptors dictate the signaling mode, i.e., excitation or inhibition, and the response dynamics of the neuron. An individual receptor can mediate both excitatory and inhibitory responses to different odorants in the same cell, suggesting a model of odorant receptor transduction. Receptors vary widely in their breadth of tuning, and odorants vary widely in the number of receptors they activate. Together, these properties provide a molecular basis for odor coding by the receptor repertoire of an olfactory organ.

Targeted mutation of a Drosophila odor receptor defines receptor requirement in a novel class of sensillum.

In vertebrates, individual olfactory neurons are thought to express a single odorant receptor (Or) gene, but it is not clear that all odor-evoked activity in each neuron is exclusively dependent on an individual odorant receptor. In Drosophila, little is known about what receptors impart odor sensitivity to particular olfactory neurons. Here, we demonstrate the use of gene targeting to produce a null mutant of the putative odorant receptor Or43b and find that the mutant is defective for odor-evoked activity in ab8A neurons, a single functional class of olfactory neurons in Drosophila. ab8A neurons lacking Or43b are still present in the mutants and display spontaneous activity but are insensitive to odor stimulation. Therefore, Or43b is required for odor responsiveness in these olfactory neurons in vivo. Or83b, a receptor expressed in a large fraction of olfactory neurons including Or43b neurons, does not confer odor responsiveness in the absence of Or43b. Olfactory behavior elicited by odorants that activate the ab8A neurons is indistinguishable between Or43b mutants and controls, demonstrating a surprising degree of functional redundancy among the limited odor receptor repertoire in this species. These studies demonstrate that a reverse genetic approach can be used to correlate specific olfactory receptors with odor specificity of functional classes of olfactory neurons.

Integrating the molecular and cellular basis of odor coding in the Drosophila antenna.

We investigate how the molecular and cellular maps of the Drosophila olfactory system are integrated. A correspondence is established between individual odor receptors, neurons, and odors. We describe the expression of the Or22a and Or22b receptor genes, show localization to dendritic membranes, and find sexual dimorphism. Or22a maps to the ab3A neuron, which responds to ethyl butyrate. Analysis of a deletion mutant lacking Or22a, along with transgenic rescue experiments, confirms the mapping and demonstrates that an Or gene is required for olfactory function in vivo. Ectopic expression of Or47a in a mutant cell identifies the neuron from which it derives and its odor ligands. Ectopic expression in a wild-type cell shows that two receptors can function in a single cell. The ab3A neuron does not depend on normal odor receptor gene expression to navigate to its target in the CNS.

Deficient pheromone responses in mice lacking a cluster of vomeronasal receptor genes.

The mammalian vomeronasal organ (VNO), a part of the olfactory system, detects pheromones--chemical signals that modulate social and reproductive behaviours. But the molecular receptors in the VNO that detect these chemosensory stimuli remain undefined. Candidate pheromone receptors are encoded by two distinct and complex superfamilies of genes, V1r and V2r (refs 3 and 4), which code for receptors with seven transmembrane domains. These genes are selectively expressed in sensory neurons of the VNO. However, there is at present no functional evidence for a role of these genes in pheromone responses. Here, using chromosome engineering technology, we delete in the germ line of mice an approximately 600-kilobase genomic region that contains a cluster of 16 intact V1r genes. These genes comprise two of the 12 described V1r gene families, and represent approximately 12% of the V1r repertoire. The mutant mice display deficits in a subset of VNO-dependent behaviours: the expression of male sexual behaviour and maternal aggression is substantially altered. Electrophysiologically, the epithelium of the VNO of such mice does not respond detectably to specific pheromonal ligands. The behavioural impairment and chemosensory deficit support a role of V1r receptors as pheromone receptors.