Molecular and cellular organization of insect chemosensory neurons

Animals use their chemosensory systems to detect and discriminate among chemical cues in the environment. Remarkable progress has recently been made in our knowledge of the molecular and cellular basis of chemosensory perception in insects, based largely on studies in Drosophila. This progress has been possible due to the identification of gene families for olfactory and gustatory receptors, the use of electro-physiological recording techniques on sensory neurons, the multitude of genetic manipulations that are available in this species, and insights from several insect model systems. Recent studies show that the superfamily of chemoreceptor proteins represent the essential elements in chemosensory coding, endowing chemosensory neurons with their abilities to respond to specific sets of odorants, tastants or pheromones. Investigating how insects detect chemicals in their environment can show us how receptor protein structures relate to ligand binding, how nervous systems process complex information, and how chemosensory systems and genes evolve.     

昆虫感觉气味的细胞与分子机制研究进展

昆虫作为地球上最为成功的类群,已经成功地进化了精细的化学感受系统,通过化学感受系统适应各种复杂的环境,保持种群的繁荣。自1991年在动物中发现嗅觉受体基因以来,关于昆虫感受化学信息的周缘神经系统的分子和细胞机制方面的进展十分迅速。文章主要就昆虫周缘神经系统的感受化学信息的分子和细胞机制进行综述。首先对昆虫感觉气味的细胞机制的研究进展进行简要介绍。昆虫嗅觉神经元在感受化学信息过程中起着极为重要的作用,昆虫嗅觉神经元上表达的嗅觉受体不同而执行着各异的功能。各种嗅觉神经元对于化学信息的感受谱有较大的区别;嗅觉神经元对化学信息类型、浓度、流动动态等产生相应的电生理特征反应。研究表明同一种神经原可以感受多种化学信息,而一种化学信息也可以被多种神经原所感受。由神经原对化学信息感受所形成的特征组合就是感受化学信息的编码。其次较为详细地论述与昆虫感受气味分子相关的一些蛋白质的研究进展。气味分子结合蛋白是一类分子量较小、水溶性的蛋白,主要位于化学感受器神经原树突周围的淋巴液中。在结构上的主要特征是具有6个保守的半光氨酸和由6个α螺旋组成的结合腔。自1981年发现以来,已经在40余种昆虫中发现上百种。由于研究手段的不断进步,已经对该类蛋白的表达特征、结合特性以及三维结构和结合位点进行了大量的研究,提出了多个可能的功能假说,在诸多的假说中,较为广泛接受的是气味分子结合蛋白在昆虫感觉气味的过程中,是与疏水性的气味分子相结合,并将气味分子运输到嗅觉神经原树突膜上的嗅觉受体上。这些处于树突膜上的嗅觉受体则是昆虫感觉气味过程中的另一个十分重要的蛋白质。目前,已经在果蝇、按蚊、蜜蜂和家蚕等10余个昆虫种类中发现上百个嗅觉受体蛋白基因。这类蛋白是跨膜蛋白,一般具有7个跨膜区,整个蛋白的氨基酸残基在400～600个。昆虫的嗅觉受体蛋白的N-端在胞内,而C-端在胞外,这与G耦联蛋白不同。而且,昆虫的一个嗅觉神经元可以表达1～3个嗅觉受体蛋白,也与哺乳动物的一个神经元只表达一种受体蛋白有所不同。每种嗅觉受体可以感受多种气味分子,而一种气味分子可以被多个嗅觉受体所感知,这样组成了感受化学信息的编码谱。最近采用基因敲除技术和膜片钳技术研究发现,昆虫的嗅觉受体蛋白在信号传导中也有特殊性,即嗅觉受体可以直接作为离子通道,而引起动作电位。还有近来的研究表明,神经膜蛋白对于果蝇的性信息素感受神经元感受性信息素cVA是必要的。实际上,昆虫对于化学信息的感受和信号的转导,并不是上述蛋白单独起作用完成的,而是多种蛋白相互作用的结果。论文最后对该领域研究内容进行了展望。     

Olfactory-based discrimination learning in the moth, Manduca sexta

Moths possess highly tuned olfactory capabilities, which can detect very low concentrations of pheromonal odorants. Much is known about the structure and function of the moth olfactory system with respect to detection of pheromones. However, we lack an understanding of the broader olfactory system, in particular, to what degree are moths capable of detecting and discriminating odorants that are not components of pheromone blends. Here we describe a methodology used to investigate the discriminability of nonpheromonal odors in moths. In a series of experiments we show that the moth Manduca sexta can (1) discriminate a number of different odors but (2) that methyl jasmonate, neither readily conditions to a food reward nor is it readily discriminated from another odor. The lack of a response to methyl jasmonate may be related to its role in host plant defense. This work provides a basis for future mapping of physiological and pharmacological studies of nonpheromonal coding in insects onto learned behavioral responses to those odorants.     

Disruptive physiology: olfaction and the microbiome–gut–brain axis

This review covers the field of olfaction and chemosensation of odorants and puts this information into the context of interactions between microbes and behaviour; the microbiome–gut–brain axis (MGBA). Recent emphasis has also been placed on the concept of the holobiome which states that no single aspect of an organism should be viewed separately and thus must include examination of their associated microbial populations and their influence. While it is known that the microbiome may be involved in the modulation of animal behaviour, there has been little systematized effort to incorporate into such studies the rapidly developing knowledge of the wide range of olfactory systems. The classical olfactory system is evolutionarily conserved in multiple taxa from insects through to fish, reptiles and mammals, and is represented by the largest gene families in vertebrates. Mice have over 1000 different olfactory receptors and humans about 400. They are distributed throughout the body and are even found in spermatozoa where they function in chemotaxis. Each olfactory receptor has the unique functional capability of high‐affinity binding to several different molecular ligands. These and other properties render the cataloguing of odorants (odorome) with specific actions a difficult task. Some ectopic olfactory receptors have been shown to have functional effects in the gut and kidney, highlighting the complexity of the systems engaged by odorants. However, there are, in addition to classical olfactory receptors, at least two other families of receptors involved in olfaction that are also widely found expressed on tissues in many different organs in addition to the nervous system and brain: the trace‐amine associated and formyl peptide receptors. Bacteria can make many if not most odorants and are responsible for recognition of species and relative relatedness, as well as predator presence, among many other examples. Activation of different combinations of olfactory receptors by bacterial products such as β‐phenylethylamine have been shown even to control expression of emotions such as fear and aggression. The number of examples of bacterial products and volatile odorants influencing brain function and behaviour is expanding rapidly. Since it is recognized that many different bacterial products and metabolites also function as social cues, and may themselves be directly or indirectly causative of behavioural change, it becomes ever more important to include olfaction into studies of the MGBA. Clearly there are broader implications for the involvement of olfaction in this rapidly evolving field. These include improvement in our understanding of the pathways engaged in various behaviours, and the identification of novel approaches and new targets in efforts to modulate behavioural changes.     

Human olfactory receptor families and their odorants

The human nose detects volatile chemical stimuli by at least three different receptor families: odorant receptors, trace amine-associated receptors, and vomeronasal type-1 receptors. As G protein-coupled receptors, all of the few functionally characterized olfactory receptors share major functional features: when expressed in heterologous cell systems, they 1) respond to odorants of certain chemical groups, e.g., amines, aliphatic carboxylic acids or aldehydes, floral or fruity odorants, including certain key-food odorants, and putative pheromones, and 2) transduce their signals to intracellular cAMP signaling. However, little is known yet about specific differences in the functional designation of the three olfactory receptor families. Recently, two heterologous cell systems expressing olfactory signaling molecules have been developed. Different screening strategies will shed light on the yet sparsely available odorant specificity profiles and structure-function relationships of olfactory receptors, as well as the structure-activity relationships of their odorants.     

Perireceptor events in olfaction

Before reaching olfactory receptor neurons, odorant molecules have to cross an aqueous interface: the nasal mucus in vertebrates and the sensillar lymph in insects. Biochemical interactions taking place between odorants and the elements of these phases are called perireceptor events. Main protein constituents of these media, in both insects and vertebrates, are OBPs (odorant-binding proteins). Another class of proteins active in the olfactory perireceptor area includes odorant-degrading enzymes. The structure and the properties of these major proteins, with particular reference to OBPs, are reviewed and their role in olfactory transduction is discussed. © 1996 John Wiley & Sons, Inc.     

Development of wiring specificity in the olfactory system

The olfactory system discriminates a large number of odorants using precisely wired neural circuits. It offers an excellent opportunity to study mechanisms of neuronal wiring specificity at the single synapse level. Each olfactory receptor neuron typically expresses only one olfactory receptor from many receptor genes (1000 in mice). In mice, this striking singularity appears to be ensured by a negative feedback mechanism. Olfactory receptor neurons expressing the same receptor converge their axons to stereotypical positions with high precision, a feature that is conserved from insects to mammals. Several molecules have recently been identified that control this process, including olfactory receptors themselves in mice. The second order neurons, mitral cells in mammals and projection neurons in insects, have a similar degree of wiring specificity: studies in Drosophila suggest that projection neuron-intrinsic mechanisms regulate their precise dendritic targeting. Finally, recent studies have revealed interactions of different cell types during circuit assembly, including axon-axon interactions among olfactory receptor neurons and dendro-dendritic interactions of projection neurons, that are essential in establishing wiring specificity of the olfactory circuit.     

Olfactory detectability of L-amino acids in the European honeybee (Apis mellifera)

The honeybee is one of several insect model systems for the study of olfaction, yet our knowledge regarding the spectrum of odorants detectable by Apis mellifera is limited. One class of odorants that has never been tested so far are the amino acids, which are important constituents of floral nectar. Using the proboscis extension response paradigm, we assessed whether the odor of amino acids is detectable for honeybees and determined olfactory detection thresholds for those amino acids that were detectable. We found that honeybees are able to detect the odor of 5 of the 20 proteinogenic amino acids when presented at a concentration of 50 or 100 mM. Median olfactory detection thresholds for these 5 amino acids were 12.5 mM with L-tyrosine and L-cysteine, 50 mM with L-tryptophan and L-asparagine, and 100 mM with L-proline. All detection thresholds were much higher than reported concentrations of amino acids in floral nectars. We conclude that in the foraging and feeding context, honeybees are likely to detect amino acids through taste rather than olfaction. Across-species comparisons of the detectability of and sensitivity to amino acids suggest that the number of functional genes coding for olfactory receptors may affect both a species' sensitivity for odorants and the breadth of its spectrum of detectable odorants.     

Olfactory Sensitivity for Six Predator Odorants in CD-1 Mice,Human Subjects,and Spider Monkeys

Using a conditioning paradigm, we assessed the olfactory sensitivity of six CD-1 mice (Mus musculus) for six sulfur-containing odorants known to be components of the odors of natural predators of the mouse. With all six odorants, the mice discriminated concentrations <0.1 ppm (parts per million) from the solvent, and with five of the six odorants the best-scoring animals were even able to detect concentrations <1 ppt (parts per trillion). Four female spider monkeys (Ateles geoffroyi) and twelve human subjects (Homo sapiens) tested in parallel were found to detect the same six odorants at concentrations <0.01 ppm, and with four of the six odorants the best-scoring animals and subjects even detected concentrations <10 ppt. With all three species, the threshold values obtained here are generally lower than (or in the lower range of) those reported for other chemical classes tested previously, suggesting that sulfur-containing odorants may play a special role in olfaction. Across-species comparisons showed that the mice were significantly more sensitive than the human subjects and the spider monkeys with four of the six predator odorants. However, the human subjects were significantly more sensitive than the mice with the remaining two odorants. Human subjects and spider monkeys significantly differed in their sensitivity with only two of the six odorants. These comparisons lend further support to the notion that the number of functional olfactory receptor genes or the relative or absolute size of the olfactory bulbs are poor predictors of a species’ olfactory sensitivity. Analysis of odor structure–activity relationships showed that in both mice and human subjects the type of alkyl rest attached to a thietane and the type of oxygen moiety attached to a thiol significantly affected olfactory sensitivity.     

Spatial representation of odorant valence in an insect brain

Brains have to decide whether and how to respond to detected stimuli based on complex sensory input. The vinegar fly Drosophila melanogaster evaluates food sources based on olfactory cues. Here, we performed a behavioral screen using the vinegar fly and established the innate valence of 110 odorants. Our analysis of neuronal activation patterns evoked by attractive and aversive odorants suggests that even though the identity of odorants is coded by the set of activated receptors, the main representation of odorant valence is formed at the output level of the antennal lobe. The topographic clustering within the antennal lobe of valence-specific output neurons resembles a corresponding domain in the olfactory bulb of mice. The basal anatomical structure of the olfactory circuit between insects and vertebrates is known to be similar; our study suggests that the representation of odorant valence is as well.     

The role of glomeruli in the neural representation of odours: results from optical recording studies

Odours are received by olfactory receptors, which send their axons to the first sensory neuropils, the antennal lobes (in insects) or the olfactory bulb (in vertebrates). From here, processed olfactory information is relayed to higher-order brain centres. A striking similarity in olfactory systems across animal phyla is the presence of glomeruli in this first sensory neuropil. Various experiments have shown that odours elicit a mosaic of activated glomeruli, suggesting that odour quality is coded in an 'across-glomeruli' activity code. In recent years, studies using optical recording techniques have greatly improved our understanding of the resulting 'across-glomeruli pattern', making it possible to simultaneously measure responses in several, often identifiable, glomeruli. For the honeybee Apis mellifera, a functional atlas of odour representation is being created: in this atlas, the glomeruli that are activated by different odorants are named. However, several limitations remain to be investigated. In this paper, we review what optical recording of odour-evoked glomerular activity patterns has revealed so far, and discuss the necessary next steps, with emphasis on the honeybee.     

Odor-guided behavior in Drosophila requires calreticulin

The efficient processing of olfactory information is crucial for many aspects of life in animals, including behavior in insects. While much is known about the organization of the insect olfactory system, comparatively little is understood about the molecules that support its function. To further elucidate the molecular basis of olfaction, we explored the role of the calcium-binding chaperone calreticulin in the behavioral response of Drosophila to aversive odorants. We show that avoidance of naturally aversive odorants is impaired in flies harboring mutations in Calreticulin. Calreticulin mutants have broad defects in odor avoidance without abnormalities in antennal responses to odorants, alterations in central nervous system structure, or deficits in overall locomotor abilities. Interestingly, Calreticulin mutants exhibit defects in behavioral responses to odorants at low strength, whereas responses to higher odorant concentrations are preserved in these animals. Our studies indicate that calreticulin plays a key role in olfactory system function, possibly by establishing its overall sensitivity to odorants.     

Molecular determinants of odorant receptor function in insects

The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later we can add computational methods to further our understanding of how specific odorants are detected by receptors. Using a comparative approach we identify two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.     

No oral-cavity-only discrimination of purely olfactory odorants

The purely olfactory odorants coumarin, octanoic acid, phenylethyl alcohol, and vanillin had been found to be consistently identified when presented retronasally but could not be identified when presented oral-cavity only (OCO). However, OCO discrimination of these odorants was not tested. Consequently, it remained possible that the oral cavity trigeminal system might provide sufficient information to differentiate these purely olfactory odorants. To evaluate this, 20 participants attempted to discriminate vapor-phase coumarin, octanoic acid, phenylethyl alcohol, and vanillin and, as a control, the trigeminal stimulus peppermint extract, from their glycerin solvent, all presented OCO. None of the purely olfactory odorants could be discriminated OCO, but, as expected, peppermint extract was consistently discriminated. This inability to discriminate clarifies and expands the previous report of lack of OCO identification of purely olfactory odorants. Taken together with prior data, these results suggest that the oral cavity trigeminal system is fully unresponsive to these odorants in vapor phase and that coumarin, octanoic acid, phenylethyl alcohol, and vanillin are indeed purely olfactory stimuli. The OCO discrimination of peppermint extract demonstrated that the absence of discrimination for the purely olfactory odorants was odorant dependent and confirmed that the oral cavity trigeminal system will provide differential response information to some vapor-phase stimuli.     

酵母双杂交筛选华北大黑鳃金龟触角均一化cDNA文库

昆虫错综复杂的嗅觉系统在昆虫寻找寄主、 交配、 产卵以及逃避行为中起到了至关重要的作用。因此, 对昆虫嗅觉感受机理的研究将有助于揭开昆虫感受和识别环境中的气味物质并引起相关行为反应的秘密。为了更多地了解华北大黑鳃金龟Holotrichia oblita Faldermann 嗅觉相关蛋白互作机制, 本实验利用DUALhunter Starter Kits酵母双杂交筛选试剂盒, 构建了华北大黑鳃金龟气味结合蛋白OBP2诱饵载体, 筛选了华北大黑鳃金龟触角酵母双杂交系统均一化的cDNA文库。通过β-半乳糖苷酶活性检测以及GenBank中Blast比对分析, 以OBP2为诱饵鉴定出6个阳性互作物。我们推测其中的一种较强的阳性互作物凝固酶原可能是华北大黑鳃金龟在嗅觉识别过程中的相关蛋白。     

Heterogeneity of molecular mechanisms the olfactor reception

In experiments on the frog isolated olfactory epithelium by using vital fluorescent microscope, odorants with fruit, rank, flower and camphor smell were shown to involve intracellular signaling systems in olfactory transduction. The odorants with different qualitative smells have different messenger and activity mechanisms. Intracellular messengers do not participate in reception of odorants with piquant and rotten smells. Thus the perception of different odour substances is maintained by physical and chemical processes. Hence, not only taste, carotid, medullar, but olfactory reception as well are characterised by heterogeneity of biophysical mechanisms.     

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection^1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination^4-6, herbivory of food crops⁷, and transmission of disease^8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)^10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed ''GCMR'') from neurons in the antennal lobe (AL; the insect''s primary olfactory center)^13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna''s receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques^14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.     

Pheromonal and host-odor processing in the insect antennal lobe: how different?

In the olfactory bulb of vertebrates and the antennal lobe of insects, precise connections between sensory receptor cells and olfactory glomeruli form the basis of a highly organized chemotopic map at the first stage of central processing in the brain. Beyond this basic level of organization, the olfactory system is typically separated into two subsystems: a 'main' olfactory pathway that detects and processes information about most environmental odorants, and an 'accessory' olfactory pathway that is devoted to information about social signals such as sex pheromones. A growing number of studies show, however, that it is not always possible to draw clear functional distinctions between the two subsystems. These findings have led some to speculate that the organizational principles by which olfactory stimuli are represented across glomeruli may be more similar in these two olfactory subsystems than previously thought.     

Modulation by octopamine of olfactory responses to nonpheromone odorants in the cockroach, Periplaneta americana L

Olfactory receptor cells in insects are modulated by neurohormones. Recordings from cockroach olfactory sensilla showed that a subset of sensory neurons increase their responses to selected nonpheromone odorants after octopamine application. With octopamine application, recordings demonstrated increased firing rates by the short but not the long alcohol-sensitive sensilla to the nonpheromone volatile, hexan-1-ol. Within the same sensillum, individual receptor cells are shown to be modulated independently from each other, indicating that the octopamine receptors reside in the receptor not in the accessory cells. A uniform decrease in the amplitude of electroantennogram, which is odorant independent, is suggested to reflect the rise in octopamine concentration in the antennal hemolymph. Perception of general odorants measured as behavioral responses changed qualitatively under octopamine treatment: namely, repulsive hexan-1-ol became neutral, whereas neutral eucalyptol became attractive. Octopamine induced a change in male behavioral responses to general odors that were essentially the same as in the state of sexual arousal. Our findings suggest that sensitivity to odors having different biological significances is modulated selectively at the peripheral as well as other levels of olfactory processing.     

Mixture and odorant processing in the olfactory systems of insects: a comparative perspective

Natural olfactory stimuli are often complex mixtures of volatiles, of which the identities and ratios of constituents are important for odor-mediated behaviors. Despite this importance, the mechanism by which the olfactory system processes this complex information remains an area of active study. In this review, we describe recent progress in how odorants and mixtures are processed in the brain of insects. We use a comparative approach toward contrasting olfactory coding and the behavioral efficacy of mixtures in different insect species, and organize these topics around four sections: (1) Examples of the behavioral efficacy of odor mixtures and the olfactory environment; (2) mixture processing in the periphery; (3) mixture coding in the antennal lobe; and (4) evolutionary implications and adaptations for olfactory processing. We also include pertinent background information about the processing of individual odorants and comparative differences in wiring and anatomy, as these topics have been richly investigated and inform the processing of mixtures in the insect olfactory system. Finally, we describe exciting studies that have begun to elucidate the role of the processing of complex olfactory information in evolution and speciation.