Elevated ozone disrupts mating boundaries in drosophilid flies.

Animals employ different strategies to establish mating boundaries between closely related species, with sex pheromones often playing a crucial role in identifying conspecific mates. Many of these pheromones have carbon-carbon double bonds, making them vulnerable to oxidation by certain atmospheric oxidant pollutants, including ozone. Here, we investigate whether increased ozone compromises species boundaries in drosophilid flies. We show that short-term exposure to increased levels of ozone degrades pheromones of Drosophila melanogaster, D. simulans, D. mauritiana, as well as D. sechellia, and induces hybridization between some of these species. As many of the resulting hybrids are sterile, this could result in local population declines. However, hybridization between D. simulans and D. mauritiana as well as D. simulans and D. sechellia results in fertile hybrids, of which some female hybrids are even more attractive to the males of the parental species. Our experimental findings indicate that ozone pollution could potentially induce breakdown of species boundaries in insects.

How conspecific and allospecific eggs and larvae drive oviposition preference in Drosophila.

Where to lay the eggs is a crucial decision for females as it influences the success of their offspring. Female flies prefer to lay eggs on food already occupied and consumed by larvae, which facilitates social feeding, but potentially could also lead to detrimental interactions between species. Whether females can modulate their attraction to cues associated with different species is unknown. Here, we analyzed the chemical profiles of eggs and larvae of 16 Drosophila species, and tested whether Drosophila flies would be attracted to larvae-treated food or food with eggs from 6 different Drosophila species. The chemical analyses revealed that larval profiles from different species are strongly overlapping, while egg profiles exhibit significant species specificity. Correspondingly, female flies preferred to lay eggs where they detected whatever species' larval cues, while we found a significant oviposition preference only for eggs of some species but not others. Our findings suggest that both larval and egg cues present at a given substrate can drive oviposition preference in female flies.

Females smell differently: characteristics and significance of the most common olfactory sensilla of female silkmoths.

In the silkmoth Bombyx mori, the role of male sensilla trichodea in pheromone detection is well established. Here we study the corresponding female sensilla, which contain two olfactory sensory neurons (OSNs) and come in two lengths, each representing a single physiological type. Only OSNs in medium trichoids respond to the scent of mulberry, the silkworm's exclusive host plant, and are more sensitive in mated females, suggesting a role in oviposition. In long trichoids, one OSN is tuned to (+)-linalool and the other to benzaldehyde and isovaleric acid, both odours emitted by silkworm faeces. While the significance of (+)-linalool detection remains unclear, isovaleric acid repels mated females and may therefore play a role in avoiding crowded oviposition sites. When we examined the underlying molecular components of neurons in female trichoids, we found non-canonical co-expression of Ir8a, the co-receptor for acid responses, and ORco, the co-receptor of odorant receptors, in long trichoids, and the unexpected expression of a specific odorant receptor in both trichoid sensillum types. In addition to elucidating the function of female trichoids, our results suggest that some accepted organizational principles of the insect olfactory system may not apply to the predominant sensilla on the antenna of female B. mori.

Odorant detection in a locust exhibits unusually low redundancy.

Olfactory coding, from insects to humans, is canonically considered to involve considerable across-fiber coding already at the peripheral level, thereby allowing recognition of vast numbers of odor compounds. We show that the migratory locust has evolved an alternative strategy built on highly specific odorant receptors feeding into a complex primary processing center in the brain. By collecting odors from food and different life stages of the locust, we identified 205 ecologically relevant odorants, which we used to deorphanize 48 locust olfactory receptors via ectopic expression in Drosophila. Contrary to the often broadly tuned olfactory receptors of other insects, almost all locust receptors were found to be narrowly tuned to one or very few ligands. Knocking out a single receptor using CRISPR abolished physiological and behavioral responses to the corresponding ligand. We conclude that the locust olfactory system, with most olfactory receptors being narrowly tuned, differs from the so-far described olfactory systems.

Evolution at multiple processing levels underlies odor-guided behavior in the genus Drosophila.

Olfaction is a fundamental sense guiding animals to their food. How the olfactory system evolves and influences behavior is still poorly understood. Here, we selected five drosophilid species, including Drosophila melanogaster, inhabiting different ecological niches to compare their olfactory systems at multiple levels. We first identified ecologically relevant natural food odorants from every species and established species-specific odorant preferences. To compare odor coding in sensory neurons, we analyzed the antennal lobe (AL) structure, generated glomerular atlases, and developed GCaMP transgenic lines for all species. Although subsets of glomeruli showed distinct tuning profiles, odorants inducing species-specific preferences were coded generally similarly. Species distantly related or occupying different habitats showed more evident differences in odor coding, and further analysis revealed that changes in olfactory receptor (OR) sequences partially explain these differences. Our results demonstrate that genetic distance in phylogeny and ecological niche occupancy are key determinants in the evolution of ORs, AL structures, odor coding, and behavior. Interestingly, changes in odor coding among species could not be explained by evolutionary changes at a single olfactory processing level but rather are a complex phenomenon based on changes at multiple levels.

Olfactory Mating Signals in the Migratory Locust Locusta migratoria.

Swarming locusts cause huge plagues across the world threatening food production. Before swarms form, locust populations exhibit a dramatic phase change from a solitary to a gregarious phase. The cause of this phase change is a complicated interplay of conspecific and environmental cues and is, especially for one of the major pests, the migratory locust Locusta migratoria, still not well understood. Here we study the behavior of both solitary and gregarious L. migratoria towards the headspace odors of conspecifics. As we do not find a general attraction of gregarious animals to the headspace of gregarious conspecifics, swarm formation does not seem to be mainly governed by olfactory aggregation cues. When testing for potential mating signals, we observe that the headspace of virgin gregarious females is highly attractive only towards virgin males of the same phase, while mated gregarious males and solitary males, regardless of their mating state, do not become attracted. Interestingly, this phase-specific attraction goes along with the finding, that mating behavior in experiments with inter-phasic pairings is extremely rare. Our data suggest that odor emissions in L. migratoria play a significant role in a mating context.

Temperature-dependent modulation of odor-dependent behavior in three drosophilid fly species of differing thermal preference.

Rapid and ongoing climate change increases global temperature, impacts feeding, and reproduction in insects. The olfaction plays an important underlying role in these behaviors in most insect species. Here, we investigated how changing temperatures affect odor detection and ensuing behavior in three drosophilid flies: Drosophila novamexicana, D. virilis and D. ezoana, species adapted to life in desert, global, and subarctic climates, respectively. Using a series of thermal preference assays, we confirmed that the three species indeed exhibit distinct temperature preferences. Next, using single sensillum recording technique, we classified olfactory sensory neurons (OSNs) present in basiconic sensilla on the antenna of the three species and thereby identified ligands for each OSN type. In a series of trap assays we proceeded to establish the behavioral valence of the best ligands and chose guaiacol, methyl salicylate and isopropyl benzoate as representatives of a repellent, attractant and neutral odor. Next, we assessed the behavioral valence of these three odors in all three species across a thermal range (10-35 °C), with flies reared at 18 °C and 25 °C. We found that both developmental and experimental temperatures affected the behavioral performance of the flies. Our study thus reveals temperature-dependent changes in odor-guided behavior in drosophilid flies.

Carbonyl products of ozone oxidation of volatile organic compounds can modulate olfactory choice behavior in insects.

Insects are a diverse group of organisms that provide important ecosystem services like pollination, pest control, and decomposition and rely on olfaction to perform these services. In the Anthropocene, increasing concentrations of oxidant pollutants such as ozone have been shown to corrupt odor-driven behavior in insects by chemically degrading e.g. flower signals or insect pheromones. The degradation, however, does not only result in a loss of signals, but also in a potential enrichment of oxidation products, predominantly small carbonyls. Whether and how these oxidation products affect insect olfactory perception remains unclear. We examined the effects of ozone-generated small carbonyls on the olfactory behavior of the vinegar fly Drosophila melanogaster. We compiled a broad collection of neurophysiologically relevant odorants for the fly from databases and literature and predicted the formation of the types of stable small carbonyl products resulting from the odorant's oxidation by ozone. Based on these predictions, we evaluated the olfactory detection and behavioral impact of the ten most frequently predicted carbonyl products in the fly using single sensillum recordings (SSRs) and behavioral tests. Our results demonstrate that the fly's olfactory system can detect the oxidation products, which then elicit either attractive or neutral behavioral responses, rather than repulsion. However, certain products alter behavioral choices to an attractive odor source of balsamic vinegar. Our findings suggest that the enrichment of small carbonyl oxidation products due to increased ozone levels can affect olfactory guided insect behavior. Our study underscores the implications for odor-guided foraging in insects and the essential ecosystem services they offer under carbonyl enriched environments.

Modulation of the NO-cGMP pathway has no effect on olfactory responses in the Drosophila antenna.

Olfaction is a crucial sensory modality in insects and is underpinned by odor-sensitive sensory neurons expressing odorant receptors that function in the dendrites as odorant-gated ion channels. Along with expression, trafficking, and receptor complexing, the regulation of odorant receptor function is paramount to ensure the extraordinary sensory abilities of insects. However, the full extent of regulation of sensory neuron activity remains to be elucidated. For instance, our understanding of the intracellular effectors that mediate signaling pathways within antennal cells is incomplete within the context of olfaction in vivo. Here, with the use of optical and electrophysiological techniques in live antennal tissue, we investigate whether nitric oxide signaling occurs in the sensory periphery of Drosophila. To answer this, we first query antennal transcriptomic datasets to demonstrate the presence of nitric oxide signaling machinery in antennal tissue. Next, by applying various modulators of the NO-cGMP pathway in open antennal preparations, we show that olfactory responses are unaffected by a wide panel of NO-cGMP pathway inhibitors and activators over short and long timescales. We further examine the action of cAMP and cGMP, cyclic nucleotides previously linked to olfactory processes as intracellular potentiators of receptor functioning, and find that both long-term and short-term applications or microinjections of cGMP have no effect on olfactory responses in vivo as measured by calcium imaging and single sensillum recording. The absence of the effect of cGMP is shown in contrast to cAMP, which elicits increased responses when perfused shortly before olfactory responses in OSNs. Taken together, the apparent absence of nitric oxide signaling in olfactory neurons indicates that this gaseous messenger may play no role as a regulator of olfactory transduction in insects, though may play other physiological roles at the sensory periphery of the antenna.

A chemical defense deters cannibalism in migratory locusts.

Many animals engage in cannibalism to supplement their diets. Among dense populations of migratory locusts, cannibalism is prevalent. We show that under crowded conditions, locusts produce an anticannibalistic pheromone called phenylacetonitrile. Both the degree of cannibalism and the production of phenylacetonitrile are density dependent and covary. We identified the olfactory receptor that detects phenylacetonitrile and used genome editing to make this receptor nonfunctional, thereby abolishing the negative behavioral response. We also inactivated the gene underlying phenylacetonitrile production and show that locusts that lack this compound lose its protection and are more frequently exposed to intraspecific predation. Thus, we reveal an anticannibalistic feature built on a specifically produced odor. The system is very likely to be of major importance in locust population ecology, and our results might therefore provide opportunities in locust management.

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei.

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally. R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with ß-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 µM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 µM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

Characterization of olfactory sensory neurons in the striped ambrosia beetle Trypodendron lineatum.

Introduction: The striped ambrosia beetle Trypodendron lineatum (Coleoptera, Curculionidae, Scolytinae) is a major forest pest in the Holarctic region. It uses an aggregation pheromone and host and non-host volatiles to locate suitable host trees, primarily stressed or dying conifer trees. The beetles bore into the xylem and inoculate spores of their obligate fungal mutualist Phialophoropsis ferruginea inside their excavated egg galleries, with the fungus serving as the main food source for the developing larvae. Olfactory sensory neuron (OSN) responses to pheromones and host volatiles are poorly understood in T. lineatum and other ambrosia beetles, and nothing is known about potential responses to fungal volatiles. Methods: We screened responses of OSNs present in 170 antennal olfactory sensilla using single sensillum recordings (SSR) and 57 odor stimuli, including pheromones, host and non-host compounds, as well as volatiles produced by P. ferruginea and fungal symbionts of other scolytine beetles. Results and Discussion: Thirteen OSN classes were characterized based on their characteristic response profiles. An OSN class responding to the aggregation pheromone lineatin was clearly the most abundant on the antennae. In addition, four OSN classes responded specifically to volatile compounds originating from the obligate fungal mutualist and three responded to non-host plant volatiles. Our data also show that T. lineatum has OSN classes tuned to pheromones of other bark beetles. Several OSN classes showed similar response profiles to those previously described in the sympatric bark beetle Ips typographus, which may reflect their shared ancestry.

Cross-generation pheromonal communication drives Drosophila oviposition site choice.

In a heterogeneous and changing environment, oviposition site selection strongly affects the survival and fitness of the offspring.1,2 Similarly, competition between larvae affects their prospects.3 However, little is known about the involvement of pheromones in regulating these processes.4,5,6,7,8 Here, we show that mated females of Drosophila melanogaster prefer to lay eggs on substrates containing extracts of conspecific larvae. After analyzing these extracts chemically, we test each compound in an oviposition assay and find that mated females display a dose-dependent preference to lay eggs on substrates spiked with (Z)-9-octadecenoic acid ethyl ester (OE). This egg-laying preference relies on gustatory receptor Gr32a and tarsal sensory neurons expressing this receptor. The concentration of OE also regulates larval place choice in a dose-dependent manner. Physiologically, OE activates female tarsal Gr32a+ neurons. In conclusion, our results reveal a cross-generation communication strategy essential for oviposition site selection and regulation of larval density.

Ozone exposure disrupts insect sexual communication.

Insect sexual communication often relies upon sex pheromones. Most insect pheromones, however, contain carbon-carbon double bonds and potentially degrade by oxidation. Here, we show that frequently reported increased levels of Anthropocenic ozone can oxidize all described male-specific pheromones of Drosophila melanogaster, resulting in reduced amounts of pheromones such as cis-Vaccenyl Acetate and (Z)-7-Tricosene. At the same time female acceptance of ozone-exposed males is significantly delayed. Interestingly, groups of ozone-exposed males also exhibit significantly increased levels of male-male courtship behaviour. When repeating similar experiments with nine other drosophilid species, we observe pheromone degradation and/or disrupted sex recognition in eight of them. Our data suggest that Anthropocenic levels of ozone can extensively oxidize double bonds in a variety of insect pheromones, thereby leading to deviations in sexual recognition.

Hedgehog-mediated gut-taste neuron axis controls sweet perception in Drosophila.

Dietary composition affects food preference in animals. High sugar intake suppresses sweet sensation from insects to humans, but the molecular basis of this suppression is largely unknown. Here, we reveal that sugar intake in Drosophila induces the gut to express and secrete Hedgehog (Hh) into the circulation. We show that the midgut secreted Hh localize to taste sensilla and suppresses sweet sensation, perception, and preference. We further find that the midgut Hh inhibits Hh signalling in the sweet taste neurons. Our electrophysiology studies demonstrate that the midgut Hh signal also suppresses bitter taste and some odour responses, affecting overall food perception and preference. We further show that the level of sugar intake during a critical window early in life, sets the adult gut Hh expression and sugar perception. Our results together reveal a bottom-up feedback mechanism involving a "gut-taste neuron axis" that regulates food sensation and preference.

Aversive Bimodal Associations Differently Impact Visual and Olfactory Memory Performance in Drosophila.

Animals form sensory associations and store them as memories to guide behavioral decisions. Although unimodal learning has been studied extensively in insects, it is important to explore sensory cues in combination because most behaviors require multimodal inputs. In our study, we optimized the T-maze to employ both visual and olfactory cues in a classical aversive learning paradigm in Drosophila melanogaster. In contrast to unimodal training, bimodal training evoked a significant short-term visual memory after a single training trial. Interestingly, the same protocol did not enhance short-term olfactory memory and even had a negative impact. However, compromised long-lasting olfactory memory significantly improved after bimodal training. Our study demonstrates that the effect of bimodal integration on learning is not always beneficial and is conditional upon the formed memory strengths. We postulate that flies utilize information on a need-to basis: bimodal training augments weakly formed memories while stronger associations are impacted differently.

Odorant receptor orthologues in conifer-feeding beetles display conserved responses to ecologically relevant odours.

Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer-feeding bark beetles Ips typographus L. ("Ityp") and Dendroctonus ponderosae Hopkins ("Dpon") (Scolytinae), and the pine weevil Hylobius abietis L. ("Habi"; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. The results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2-phenylethanol (2-PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2-PE, suggesting a shared evolutionary history of 2-PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer-feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm nonhost plants, and 2-PE being important for intraspecific communication and/or playing a putative role in beetle-microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs.

Functional olfactory evolution in Drosophila suzukii and the subgenus Sophophora.

Comparative analyses of multiple genomes are used extensively to examine the gains and losses of chemosensory receptors across the genus Drosophila. However, few studies have delved into functional olfactory characteristics. Here we assess olfactory function across 20 species, and identify and describe several similar elements of evolution. We document (a) minor changes in functional ligands based on amino acid substitutions, (b) major changes in olfactory function or perhaps entire receptor replacements, and (c) that only a few receptors are subject to repeated changes, whereas 32 out of 37 OSNs are largely functionally conserved. In addition, we generate a robust model for identifying olfactory function using genomic data and comprehensive ligand-receptor combinations, which includes the prediction of binding pockets. Moreover, this study highlights that functional olfactory evolution does not affect all chemosensory receptors equally, and that ecological, evolutionary, and developmental forces repeatedly affect only a small subset of available receptor proteins.

Higher-order olfactory neurons in the lateral horn support odor valence and odor identity coding in Drosophila.

Understanding neuronal representations of odor-evoked activities and their progressive transformation from the sensory level to higher brain centers features one of the major aims in olfactory neuroscience. Here, we investigated how odor information is transformed and represented in higher-order neurons of the lateral horn, one of the higher olfactory centers implicated in determining innate behavior, using Drosophila melanogaster. We focused on a subset of third-order glutamatergic lateral horn neurons (LHNs) and characterized their odor coding properties in relation to their presynaptic partner neurons, the projection neurons (PNs) by two-photon functional imaging. We show that odors evoke reproducible, stereotypic, and odor-specific response patterns in LHNs. Notably, odor-evoked responses in these neurons are valence-specific in a way that their response amplitude is positively correlated with innate odor preferences. We postulate that this valence-specific activity is the result of integrating inputs from multiple olfactory channels through second-order neurons. GRASP and micro-lesioning experiments provide evidence that glutamatergic LHNs obtain their major excitatory input from uniglomerular PNs, while they receive an odor-specific inhibition through inhibitory multiglomerular PNs. In summary, our study indicates that odor representations in glutamatergic LHNs encode hedonic valence and odor identity and primarily retain the odor coding properties of second-order neurons.