Ring-shaped odor coding in the antennal lobe of migratory locusts.

The representation of odors in the locust antennal lobe with its >2,000 glomeruli has long remained a perplexing puzzle. We employed the CRISPR-Cas9 system to generate transgenic locusts expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon functional imaging, we mapped the spatial activation patterns representing a wide range of ecologically relevant odors across all six developmental stages. Our findings reveal a functionally ring-shaped organization of the antennal lobe composed of specific glomerular clusters. This configuration establishes an odor-specific chemotopic representation by encoding different chemical classes and ecologically distinct odors in the form of glomerular rings. The ring-shaped glomerular arrangement, which we confirm by selective targeting of OR70a-expressing sensory neurons, occurs throughout development, and the odor-coding pattern within the glomerular population is consistent across developmental stages. Mechanistically, this unconventional spatial olfactory code reflects the locust-specific and multiplexed glomerular innervation pattern of the antennal lobe.

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.

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.

Mixing things up! - how odor blends are processed in Drosophila.

Insects have to navigate a complex and rich olfactory environment consisting of mixtures of odors at varying ratios. However, we understand little of how the olfactory system represents these complex blends. This review aims to highlight some of the recent results of studying this mixture code, in the Drosophila melanogaster olfactory system, as well as gives a short background to one of the most challenging questions in olfaction - how are mixtures encoded in the brain?

An Assassin's Secret: Multifunctional Cytotoxic Compounds in the Predation Venom of the Assassin Bug Psytalla horrida (Reduviidae, Hemiptera).

Predatory assassin bugs produce venomous saliva that enables them to overwhelm, kill, and pre-digest large prey animals. Venom from the posterior main gland (PMG) of the African assassin bug Psytalla horrida has strong cytotoxic effects, but the responsible compounds are yet unknown. Using cation-exchange chromatography, we fractionated PMG extracts from P. horrida and screened the fractions for toxicity. Two venom fractions strongly affected insect cell viability, bacterial growth, erythrocyte integrity, and intracellular calcium levels in Drosophila melanogaster olfactory sensory neurons. LC-MS/MS analysis revealed that both fractions contained gelsolin, redulysins, S1 family peptidases, and proteins from the uncharacterized venom protein family 2. Synthetic peptides representing the putative lytic domain of redulysins had strong antimicrobial activity against Escherichia coli and/or Bacillus subtilis but only weak toxicity towards insect or mammalian cells, indicating a primary role in preventing the intake of microbial pathogens. In contrast, a recombinant venom protein family 2 protein significantly reduced insect cell viability but exhibited no antibacterial or hemolytic activity, suggesting that it plays a role in prey overwhelming and killing. The results of our study show that P. horrida secretes multiple cytotoxic compounds targeting different organisms to facilitate predation and antimicrobial defense.

Experience-dependent plasticity in the olfactory system of Drosophila melanogaster and other insects.

It is long known that the nervous system of vertebrates can be shaped by internal and external factors. On the other hand, the nervous system of insects was long assumed to be stereotypic, although evidence for plasticity effects accumulated for several decades. To cover the topic comprehensively, this review recapitulates the establishment of the term "plasticity" in neuroscience and introduces its original meaning. We describe the basic composition of the insect olfactory system using Drosophila melanogaster as a representative example and outline experience-dependent plasticity effects observed in this part of the brain in a variety of insects, including hymenopterans, lepidopterans, locusts, and flies. In particular, we highlight recent advances in the study of experience-dependent plasticity effects in the olfactory system of D. melanogaster, as it is the most accessible olfactory system of all insect species due to the genetic tools available. The partly contradictory results demonstrate that morphological, physiological and behavioral changes in response to long-term olfactory stimulation are more complex than previously thought. Different molecular mechanisms leading to these changes were unveiled in the past and are likely responsible for this complexity. We discuss common problems in the study of experience-dependent plasticity, ways to overcome them, and future directions in this area of research. In addition, we critically examine the transferability of laboratory data to natural systems to address the topic as holistically as possible. As a mechanism that allows organisms to adapt to new environmental conditions, experience-dependent plasticity contributes to an animal's resilience and is therefore a crucial topic for future research, especially in an era of rapid environmental changes.

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.

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.

Shedding Light on Inter-Individual Variability of Olfactory Circuits in Drosophila.

Inter-individual differences in behavioral responses, anatomy or functional properties of neuronal populations of animals having the same genotype were for a long time disregarded. The majority of behavioral studies were conducted at a group level, and usually the mean behavior of all individuals was considered. Similarly, in neurophysiological studies, data were pooled and normalized from several individuals. This approach is mostly suited to map and characterize stereotyped neuronal properties between individuals, but lacks the ability to depict inter-individual variability regarding neuronal wiring or physiological characteristics. Recent studies have shown that behavioral biases and preferences to olfactory stimuli can vary significantly among individuals of the same genotype. The origin and the benefit of these diverse "personalities" is still unclear and needs to be further investigated. A perspective taken into account the inter-individual differences is needed to explore the cellular mechanisms underlying this phenomenon. This review focuses on olfaction in the vinegar fly Drosophila melanogaster and summarizes previous and recent studies on odor-guided behavior and the underlying olfactory circuits in the light of inter-individual variability. We address the morphological and physiological variabilities present at each layer of the olfactory circuitry and attempt to link them to individual olfactory behavior. Additionally, we discuss the factors that might influence individuality with regard to olfactory perception.

Multimodal Information Processing and Associative Learning in the Insect Brain.

The study of sensory systems in insects has a long-spanning history of almost an entire century. Olfaction, vision, and gustation are thoroughly researched in several robust insect models and new discoveries are made every day on the more elusive thermo- and mechano-sensory systems. Few specialized senses such as hygro- and magneto-reception are also identified in some insects. In light of recent advancements in the scientific investigation of insect behavior, it is not only important to study sensory modalities individually, but also as a combination of multimodal inputs. This is of particular significance, as a combinatorial approach to study sensory behaviors mimics the real-time environment of an insect with a wide spectrum of information available to it. As a fascinating field that is recently gaining new insight, multimodal integration in insects serves as a fundamental basis to understand complex insect behaviors including, but not limited to navigation, foraging, learning, and memory. In this review, we have summarized various studies that investigated sensory integration across modalities, with emphasis on three insect models (honeybees, ants and flies), their behaviors, and the corresponding neuronal underpinnings.

Correction: Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi.

[This corrects the article DOI: 10.1371/journal.pntd.0009098.].

An ammonium transporter is a non-canonical olfactory receptor for ammonia.

Numerous hematophagous insects are attracted to ammonia, a volatile released in human sweat and breath.1-3 Low levels of ammonia also attract non-biting insects such as the genetic model organism Drosophila melanogaster and several species of agricultural pests.4,5 Two families of ligand-gated ion channels function as olfactory receptors in insects,6-10 and studies have linked ammonia sensitivity to a particular olfactory receptor in Drosophila.5,11,12 Given the widespread importance of ammonia to insect behavior, it is surprising that the genomes of most insects lack an ortholog of this gene.6 Here, we show that canonical olfactory receptors are not necessary for responses to ammonia in Drosophila. Instead, we demonstrate that a member of the ancient electrogenic ammonium transporter family, Amt, is likely a new type of olfactory receptor. We report two hitherto unidentified olfactory neuron populations that mediate neuronal and behavioral responses to ammonia in Drosophila. Their endogenous ammonia responses are lost in Amt mutant flies, and ectopic expression of either Drosophila or Anopheles Amt confers ammonia sensitivity. These results suggest that Amt is the first transporter known to function as an olfactory receptor in animals and that its function may be conserved across insect species.

Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi.

American trypanosomiasis, or Chagas disease, is transmitted by both domestic and sylvatic species of Triatominae which use sensory cues to locate their vertebrate hosts. Among them, odorants have been shown to play a key role. Previous work revealed morphological differences in the sensory apparatus of different species of Triatomines, but to date a comparative functional study of the olfactory system is lacking. After examining the antennal sensilla with scanning electronic microscopy (SEM), we compared olfactory responses of Rhodnius prolixus and the sylvatic Rhodnius brethesi using an electrophysiological approach. In electroantennogram (EAG) recordings, we first showed that the antenna of R. prolixus is highly responsive to carboxylic acids, compounds found in their habitat and the headspace of their vertebrate hosts. We then compared responses from olfactory sensory neurons (OSNs) housed in the grooved peg sensilla of both species, as these are tuned to these compounds using single-sensillum recordings (SSRs). In R. prolixus, the SSR responses revealed a narrower tuning breath than its sylvatic sibling, with the latter showing responses to a broader range of chemical classes. Additionally, we observed significant differences between these two species in their response to particular volatiles, such as amyl acetate and butyryl chloride. In summary, the closely related, but ecologically differentiated R. prolixus and R. brethesi display distinct differences in their olfactory functions. Considering the ongoing rapid destruction of the natural habitat of sylvatic species and the likely shift towards environments shaped by humans, we expect that our results will contribute to the design of efficient vector control strategies in the future.

Olfactory processing in the lateral horn of Drosophila.

Sensing olfactory signals in the environment represents a crucial and significant task of sensory systems in almost all organisms to facilitate survival and reproduction. Notably, the olfactory system of diverse animal phyla shares astonishingly many fundamental principles with regard to anatomical and functional properties. Binding of odor ligands by chemosensory receptors present in the olfactory peripheral organs leads to a neuronal activity that is conveyed to first and higher-order brain centers leading to a subsequent odor-guided behavioral decision. One of the key centers for integrating and processing innate olfactory behavior is the lateral horn (LH) of the protocerebrum in insects. In recent years the LH of Drosophila has garnered increasing attention and many studies have been dedicated to elucidate its circuitry. In this review we will summarize the recent advances in mapping and characterizing LH-specific cell types, their functional properties with respect to odor tuning, their neurotransmitter profiles, their connectivity to pre-synaptic and post-synaptic partner neurons as well as their impact for olfactory behavior as known so far.

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.

Everyone on Their Own! Individualization of Synaptic Boutons.

Animals associate different odors with good and bad memories. This memory formation requires high neuronal plasticity. In this issue of Neuron, Bilz et al. (2020) demonstrate that associative learning modulates individual synaptic boutons of the intrinsic neurons of the memory center of Drosophila.

Odor-Induced Multi-Level Inhibitory Maps in Drosophila.

Optical imaging of intracellular Ca2+ influx as a correlate of neuronal excitation represents a standard technique for visualizing spatiotemporal activity of neuronal networks. However, the information-processing properties of single neurons and neuronal circuits likewise involve inhibition of neuronal membrane potential. Here, we report spatially resolved optical imaging of odor-evoked inhibitory patterns in the olfactory circuitry of Drosophila using a genetically encoded fluorescent Cl- sensor. In combination with the excitatory component reflected by intracellular Ca2+ dynamics, we present a comprehensive functional map of both odor-evoked neuronal activation and inhibition at different levels of olfactory processing. We demonstrate that odor-evoked inhibition carried by Cl- influx is present both in sensory neurons and second-order projection neurons (PNs), and is characterized by stereotypic, odor-specific patterns. Cl--mediated inhibition features distinct dynamics in different neuronal populations. Our data support a dual role of inhibitory neurons in the olfactory system: global gain control across the neuronal circuitry and glomerulus-specific inhibition to enhance neuronal information processing.

Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells.

Insect odorant receptors (ORs) show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem, we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor coreceptor (Orco) and an odor-binding OR, simultaneously. We show that this new method can be used to reliably express insect ORs in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms.