Plasticity and modulation of olfactory circuits in insects.

Olfactory circuits change structurally and physiologically during development and adult life. This allows insects to respond to olfactory cues in an appropriate and adaptive way according to their physiological and behavioral state, and to adapt to their specific abiotic and biotic natural environment. We highlight here findings on olfactory plasticity and modulation in various model and non-model insects with an emphasis on moths and social Hymenoptera. Different categories of plasticity occur in the olfactory systems of insects. One type relates to the reproductive or feeding state, as well as to adult age. Another type of plasticity is context-dependent and includes influences of the immediate sensory and abiotic environment, but also environmental conditions during postembryonic development, periods of adult behavioral maturation, and short- and long-term sensory experience. Finally, plasticity in olfactory circuits is linked to associative learning and memory formation. The vast majority of the available literature summarized here deals with plasticity in primary and secondary olfactory brain centers, but also peripheral modulation is treated. The described molecular, physiological, and structural neuronal changes occur under the influence of neuromodulators such as biogenic amines, neuropeptides, and hormones, but the mechanisms through which they act are only beginning to be analyzed.

Behavioral and Antennal Responses of Tribolium confusum to Varronia globosa Essential Oil and Its Main Constituents: Perspective for Their Use as Repellent.

Essential oils of aromatic plants represent an alternative to classical pest control with synthetic chemicals. They are especially promising for the alternative control of stored product pest insects. Here, we tested behavioral and electrophysiological responses of the stored product pest Tribolium confusum, to the essential oil of a Brazilian indigenous plant, Varronia globosa, collected in the Caatinga ecosystem. We analyzed the essential oil by GC-MS, tested the effects of the entire oil and its major components on the behavior of individual beetles in a four-way olfactometer, and investigated responses to these stimuli in electroantennogram recordings (EAG). We could identify 25 constituents in the essential oil of V. globosa, with anethole, caryophyllene and spathulenole as main components. The oil and its main component anethole had repellent effects already at low doses, whereas caryophyllene had only a repellent effect at a high dose. In addition, the essential oil abolished the attractive effect of the T. confusum aggregation pheromone. EAG recordings revealed dose-dependent responses to the individual components and increasing responses to the blend and even more to the entire oil. Our study reveals the potential of anethole and the essential oil of V. globosa in the management of stored product pests.

A plant volatile alters the perception of sex pheromone blend ratios in a moth.

Mate finding in most moths is based on male perception of a female-emitted pheromone whose species specificity resides in component chemistry and proportions. Components are individually detected by specialized olfactory receptor neurons (ORNs) projecting into the macroglomerular complex (MGC) of the male brain. We asked how robust ratio recognition is when challenged by a plant volatile background. To test this, we investigated the perception of the pheromone blend in Agrotis ipsilon, a moth species whose females produce a blend of Z7-dodecenyl acetate (Z7-12:Ac), Z9-tetradecenyl acetate (Z9-14:Ac), and Z11-hexadecenyl acetate in a 4:1:4 ratio optimally attractive for males. First, we recorded the responses of specialist ORNs for Z7 and Z9 and showed that heptanal, a flower volatile, activated Z7 but not Z9 neurons. Then, we recorded intracellularly the responses of MGC neurons to various ratios and showed that heptanal altered ratio responses of pheromone-sensitive neurons. Finally, we analyzed the behavior of males in a wind tunnel and showed that their innate preference for the 4:1:4 blend was shifted in the presence of heptanal. Pheromone ratio recognition may thus be altered by background odorants. Therefore, the olfactory environment might be a selective force for the evolution of pheromone communication systems.

Plasticity in Insect Olfaction: To Smell or Not to Smell?

In insects, olfaction plays a crucial role in many behavioral contexts, such as locating food, sexual partners, and oviposition sites. To successfully perform such behaviors, insects must respond to chemical stimuli at the right moment. Insects modulate their olfactory system according to their physiological state upon interaction with their environment. Here, we review the plasticity of behavioral responses to different odor types according to age, feeding state, circadian rhythm, and mating status. We also summarize what is known about the underlying neural and endocrinological mechanisms, from peripheral detection to central nervous integration, and cover neuromodulation from the molecular to the behavioral level. We describe forms of olfactory plasticity that have contributed to the evolutionary success of insects and have provided them with remarkable tools to adapt to their ever-changing environment.

Low doses of a neonicotinoid insecticide modify pheromone response thresholds of central but not peripheral olfactory neurons in a pest insect.

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids, leaving residues in the environment. There is now evidence that low doses of insecticides can have positive effects on pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction, and olfactory synaptic transmission is cholinergic, neonicotinoid residues could modify chemical communication. We recently showed that treatments with different sublethal doses of clothianidin could either enhance or decrease behavioural sex pheromone responses in the male moth, Agrotis ipsilon. We investigated now effects of the behaviourally active clothianidin doses on the sensitivity of the peripheral and central olfactory system. We show with extracellular recordings that both tested clothianidin doses do not influence pheromone responses in olfactory receptor neurons. Similarly, in vivo optical imaging does not reveal any changes in glomerular response intensities to the sex pheromone after clothianidin treatments. The sensitivity of intracellularly recorded antennal lobe output neurons, however, is upregulated by a lethal dose 20 times and downregulated by a dose 10 times lower than the lethal dose 0. This correlates with the changes of behavioural responses after clothianidin treatment and suggests the antennal lobe as neural substrate involved in clothianidin-induced behavioural changes.

Brief sensory experience differentially affects the volume of olfactory brain centres in a moth.

Experience modifies behaviour in animals so that they adapt to their environment. In male noctuid moths, Spodoptera littoralis, brief pre-exposure to various behaviourally relevant sensory signals modifies subsequent behaviour towards the same or different sensory modalities. Correlated with a behavioural increase in responses of male moths to the female-emitted sex pheromone after pre-exposure to olfactory, acoustic or gustatory stimuli, an increase in sensitivity of olfactory neurons within the primary olfactory centre, the antennal lobe, is found for olfactory and acoustic stimuli, but not for gustatory stimuli. Here, we investigated whether anatomical changes occurring in the antennal lobes and in the mushroom bodies (the secondary olfactory centres) possibly correlated with the changes observed in behaviour and in olfactory neuron physiology. Our results showed that significant volume changes occurred in glomeruli (olfactory units) responsive to sex pheromone following exposure to both pheromone and predator sounds. The volume of the mushroom body input region (calyx) also increased significantly after pheromone and predator sound treatment. However, we found no changes in the volume of antennal lobe glomeruli or of the mushroom body calyx after pre-exposure to sucrose. These findings show a relationship of antennal lobe sensitivity changes to the pheromone with changes in the volume of the related glomeruli and the output area of antennal lobe projection neurons elicited by sensory cues causing a behavioural change. Behavioural changes observed after sucrose pre-exposure must originate from changes in higher integration centres in the brain.

Heterogeneity and convergence of olfactory first-order neurons account for the high speed and sensitivity of second-order neurons.

In the olfactory system of male moths, a specialized subset of neurons detects and processes the main component of the sex pheromone emitted by females. It is composed of several thousand first-order olfactory receptor neurons (ORNs), all expressing the same pheromone receptor, that contact synaptically a few tens of second-order projection neurons (PNs) within a single restricted brain area. The functional simplicity of this system makes it a favorable model for studying the factors that contribute to its exquisite sensitivity and speed. Sensory information--primarily the identity and intensity of the stimulus--is encoded as the firing rate of the action potentials, and possibly as the latency of the neuron response. We found that over all their dynamic range, PNs respond with a shorter latency and a higher firing rate than most ORNs. Modelling showed that the increased sensitivity of PNs can be explained by the ORN-to-PN convergent architecture alone, whereas their faster response also requires cell-to-cell heterogeneity of the ORN population. So, far from being detrimental to signal detection, the ORN heterogeneity is exploited by PNs, and results in two different schemes of population coding based either on the response of a few extreme neurons (latency) or on the average response of many (firing rate). Moreover, ORN-to-PN transformations are linear for latency and nonlinear for firing rate, suggesting that latency could be involved in concentration-invariant coding of the pheromone blend and that sensitivity at low concentrations is achieved at the expense of precise encoding at high concentrations.

Pheromone modulates plant odor responses in the antennal lobe of a moth.

In nature, male moths are exposed to a complex plant odorant environment when they fly upwind to a sex pheromone source in their search for mates. Plant odors have been shown to affect responses to pheromone at various levels but how does pheromone affects plant odor perception? We recorded responses from neurons within the non-pheromonal "ordinary glome ruli" of the primary olfactory center, the antennal lobe (AL), to single and pulsed stimulations with the plant odorant heptanal, the pheromone, and their mixture in the male moth Agrotis ipsilon. We identified 3 physiological types of neurons according to their activity patterns combining excitatory and inhibitory phases. Both local and projection neurons were identified in each physiological type. Neurons with excitatory responses to heptanal responded also frequently to the pheromone and showed additive responses to the mixture. Moreover, the neuron's ability of resolving successive pulses generally improved with the mixture. Only some neurons with combined excitatory/inhibitory, or purely inhibitory responses to heptanal, also responded to the pheromone. Although individual mixture responses were not significantly different from heptanal responses in these neurons, pulse resolution was improved with the mixture as compared with heptanal alone. These results demonstrate that the pheromone and the general odorant subsystems interact more intensely in the moth AL than previously thought.

Brief predator sound exposure elicits behavioral and neuronal long-term sensitization in the olfactory system of an insect.

Modulation of sensitivity to sensory cues by experience is essential for animals to adapt to a changing environment. Sensitization and adaptation to signals of the same modality as a function of experience have been shown in many cases, and some of the neurobiological mechanisms underlying these processes have been described. However, the influence of sensory signals on the sensitivity of a different modality is largely unknown. In males of the noctuid moth, Spodoptera littoralis, the sensitivity to the female-produced sex pheromone increases 24 h after a brief preexposure with pheromone at the behavioral and central nervous level. Here we show that this effect is not confined to the same sensory modality: the sensitivity of olfactory neurons can also be modulated by exposure to a different sensory stimulus, i.e., a pulsed stimulus mimicking echolocating sounds from attacking insectivorous bats. We tested responses of preexposed male moths in a walking bioassay and recorded from neurons in the primary olfactory center, the antennal lobe. We show that brief exposure to a bat call, but not to a behaviorally irrelevant tone, increases the behavioral sensitivity of male moths to sex pheromone 24 h later in the same way as exposure to the sex pheromone itself. The observed behavioral modification is accompanied by an increase in the sensitivity of olfactory neurons in the antennal lobe. Our data provide thus evidence for cross-modal experience-dependent plasticity not only on the behavioral level, but also on the central nervous level, in an insect.

Sensory pathway in aquatic basal polyneoptera: Antennal sensilla and brain morphology in stoneflies.

Aquatic insects represent a great portion of Arthropod diversity and the major fauna in inland waters. The sensory biology and neuroanatomy of these insects are, however, poorly investigated. This research aims to describe the antennal sensilla of nymphs of the stonefly Dinocras cephalotes using scanning electron microscopy and comparing them with the adult sensilla. Besides, central antennal pathways in nymphs and adults are investigated by neuron mass-tracing with tetramethylrhodamine, and their brain structures are visualized with an anti-synapsin antibody. No dramatic changes occur in the antennal sensilla during nymphal development, while antennal sensilla profoundly change from nymphs to adults when switching from an aquatic to an aerial lifestyle. However, similar brain structures are used in nymphs and adults to process diverging sensory information, perceived through different sensilla in water and air. These data provide valuable insights into the evolution of aquatic heterometabolous insects, maintaining a functional sensory system throughout development, including a distinct adaptation of the peripheral olfactory systems during the transition from detection of water-soluble chemicals to volatile compounds in the air. From a conservation biology perspective, the present data contribute to a better knowledge of the biology of stoneflies, which are very important bioindicators in rivers.

Function and central projections of gustatory receptor neurons on the antenna of the noctuid moth Spodoptera littoralis.

Chemosensory information is crucial for most insects to feed and reproduce. Olfactory signals are mainly used at a distance, whereas gustatory stimuli play an important role when insects directly contact chemical substrates. In noctuid moths, although the antennae are the main olfactory organ, they also bear taste sensilla. These taste sensilla detect sugars and hence are involved in appetitive learning but could also play an important role in food evaluation by detecting salts and bitter substances. To investigate this, we measured the responses of individual taste sensilla on the antennae of Spodoptera littoralis to sugars and salts using tip recordings. We also traced the projections of their neuronal axons into the brain. In each sensillum, we found one or two neurons responding to sugars: one NaCl-responsive and one water-sensitive neuron. Responses of these neurons were dose-dependent and similar across different locations on the antenna. Responses were dependent on the sex for sucrose and on both sex and location for glucose and fructose. We did not observe a spatial map for the projections from specific regions of the antennae to the deutocerebrum or the tritocerebrum/suboesophageal ganglion complex. In accordance with physiological recordings, back-fills from individual sensilla revealed up to four axons, in most cases targeting different projection zones.

Sex- and maturity-dependent antennal detection of host plant volatiles in the cabbage root fly, Delia radicum.

Adult insect behaviour in response to plant-emitted volatile compounds varies between the sexes and as a function of maturity. These differences in behavioural responses can be due to modulation in the peripheral or central nervous system. In the cabbage root fly, Delia radicum, behavioural effects of certain host plant volatiles on mature female behaviour have been evaluated, and a large number of compounds emitted by brassicaceous host plants have been identified. We recorded here dose-dependent electroantennogram responses to all tested compounds and investigated if the antennal detection of individual volatile compounds emitted by intact and damaged host plants differs between male and female, as well as immature and mature flies. Our results showed dose-dependent responses in mature and immature males and females. Mean response amplitudes varied significantly between sexes for three compounds, and between maturity states for six compounds. For some additional compounds significant differences occurred only for high stimulus doses (interaction between dose and sex and/or dose and maturity status). Multivariate analysis revealed a significant global effect of maturity on electroantennogram response amplitudes and for one experimental session also a significant global effect of the sex. Interestingly, allyl isothiocyanate, a compound stimulating oviposition behaviour, elicited stronger responses in mature than in immature flies, whereas ethylacetophenone, an attractive flower volatile, elicited stronger responses in immature than in mature flies, which correlates with the behavioural role of these compounds. Several host-derived compounds elicited stronger responses in females than in males and, at least at high doses, stronger responses in mature than in immature flies, indicating differential antennal sensitivity to behaviourally active compounds. Six compounds did not cause any significant differences in responses between the different groups of flies. Our results thus confirm peripheral plasticity in plant volatile detection in the cabbage root fly and provide a basis for future behavioural investigations on the function of individual plant compounds.

Experience-dependent modulation of antennal sensitivity and input to antennal lobes in male moths (Spodoptera littoralis) pre-exposed to sex pheromone.

Sex pheromones are intraspecific olfactory signals emitted by one sex to attract a potential mating partner. Behavioural responses to sex pheromones are generally highly stereotyped. However, they can be modulated by experience, as male moths previously exposed to female sex pheromone respond with a lower threshold upon further detection, even after long delays. Here, we address the question of the neural mechanisms underlying such long-term modulation. As previous work has shown increased responses to pheromone in central olfactory neurons, we asked whether brief exposure to the pheromone increases input activity from olfactory receptor neurons. Males pre-exposed to sex pheromone exhibited increased peripheral sensitivity to the main pheromone component. Among nine antennal genes targeted as putatively involved in pheromone reception, one encoding a pheromone-binding protein showed significant upregulation upon exposure. In the primary olfactory centre (antennal lobe), the neural compartment processing the main pheromone component was enlarged after a brief pheromone exposure, thus suggesting enduring structural changes. We hypothesise that higher peripheral sensitivity following pre-exposure leads to increased input to the antennal lobe, thus contributing to the structural and functional reorganization underlying a stable change in behaviour.

Plant odour stimuli reshape pheromonal representation in neurons of the antennal lobe macroglomerular complex of a male moth.

Male moths are confronted with complex odour mixtures in a natural environment when flying towards a female-emitted sex pheromone source. Whereas synergistic effects of sex pheromones and plant odours have been observed at the behavioural level, most investigations at the peripheral level have shown an inhibition of pheromone responses by plant volatiles, suggesting a potential role of the central nervous system in reshaping the peripheral information. We thus investigated the interactions between sex pheromone and a behaviourally active plant volatile, heptanal, and their effects on responses of neurons in the pheromone-processing centre of the antennal lobe, the macroglomerular complex, in the moth Agrotis ipsilon. Our results show that most of these pheromone-sensitive neurons responded to the plant odour. Most neurons responded to the pheromone with a multiphasic pattern and were anatomically identified as projection neurons. They responded either with excitation or pure inhibition to heptanal, and the response to the mixture pheromone + heptanal was generally weaker than to the pheromone alone, showing a suppressive effect of heptanal. However, these neurons responded with a better resolution to pulsed stimuli. The other neurons with either purely excitatory or inhibitory responses to all three stimuli did not exhibit significant differences in responses between stimuli. Although the suppression of the pheromone responses in AL neurons by the plant odour is counter-intuitive at first glance, the observed better resolution of pulsed stimuli is probably more important than high sensitivity to the localization of a calling female.

Plasticity in Chemical Host Plant Recognition in Herbivorous Insects and Its Implication for Pest Control.

Chemical communication is very important in herbivorous insects, with many species being important agricultural pests. They often use olfactory cues to find their host plants at a distance and evaluate their suitability upon contact with non-volatile cues. Responses to such cues are modulated through interactions between various stimuli of biotic and abiotic origin. In addition, the response to the same stimulus can vary as a function of, for example, previous experience, age, mating state, sex, and morph. Here we summarize recent advances in the understanding of plant localization and recognition in herbivorous insects with a focus on the interplay between long- and short-range signals in a complex environment. We then describe recent findings illustrating different types of plasticity in insect plant choice behavior and the underlying neuronal mechanisms at different levels of the chemosensory pathway. In the context of strong efforts to replace synthetic insecticides with alternative pest control methods, understanding combined effects between long- and close-range chemical cues in herbivore-plant interactions and their complex environment in host choice are crucial to develop effective plant protection methods. Furthermore, plasticity of behavioral and neuronal responses to chemical cues needs to be taken into account to develop effective sustainable pest insect control through behavioral manipulation.

Pheromone Receptor Knock-Out Affects Pheromone Detection and Brain Structure in a Moth.

Sex pheromone receptors are crucial in insects for mate finding and contribute to species premating isolation. Many pheromone receptors have been functionally characterized, especially in moths, but loss of function studies are rare. Notably, the potential role of pheromone receptors in the development of the macroglomeruli in the antennal lobe (the brain structures processing pheromone signals) is not known. Here, we used CRISPR-Cas9 to knock-out the receptor for the major component of the sex pheromone of the noctuid moth Spodoptera littoralis, and investigated the resulting effects on electrophysiological responses of peripheral pheromone-sensitive neurons and on the structure of the macroglomeruli. We show that the inactivation of the receptor specifically affected the responses of the corresponding antennal neurons did not impact the number of macroglomeruli in the antennal lobe but reduced the size of the macroglomerulus processing input from neurons tuned to the main pheromone component. We suggest that this mutant neuroanatomical phenotype results from a lack of neuronal activity due to the absence of the pheromone receptor and potentially reduced neural connectivity between peripheral and antennal lobe neurons. This is the first evidence of the role of a moth pheromone receptor in macroglomerulus development and extends our knowledge of the different functions odorant receptors can have in insect neurodevelopment.

Mating-induced differential coding of plant odour and sex pheromone in a male moth.

Innate behaviours in animals can be influenced by several factors, such as the environment, experience, or physiological status. This behavioural plasticity originates from changes in the underlying neuronal substrate. A well-described form of plasticity is induced by mating. In both vertebrates and invertebrates, males experience a post-ejaculatory refractory period, during which they avoid new females. In the male moth Agrotis ipsilon, mating induces a transient inhibition of responses to the female-produced sex pheromone. To understand the neural bases of this inhibition and its possible odour specificity, we carried out a detailed analysis of the response characteristics of the different neuron types from the periphery to the central level. We examined the response patterns of pheromone-sensitive and plant volatile-sensitive neurons in virgin and mated male moths. By using intracellular recordings, we showed that mating changes the response characteristics of pheromone-sensitive antennal lobe (AL) neurons, and thus decreases their sensitivity to sex pheromone. Individual olfactory receptor neuron (ORN) recordings and calcium imaging experiments indicated that pheromone sensory input remains constant. On the other hand, calcium responses to non-pheromonal odours (plant volatiles) increased after mating, as reflected by increased firing frequencies of plant-sensitive AL neurons, although ORN responses to heptanal remained unchanged. We suggest that differential processing of pheromone and plant odours allows mated males to transiently block their central pheromone detection system, and increase non-pheromonal odour detection in order to efficiently locate food sources.

Revisiting the anatomy of the central nervous system of a hemimetabolous model insect species: the pea aphid Acyrthosiphon pisum.

Aphids show a marked phenotypic plasticity, producing asexual or sexual and winged or wingless morphs depending on environmental conditions and season. We describe here the general structure of the brain of various morphs of the pea aphid Acyrthosiphon pisum. This is the first detailed anatomical study of the central nervous system of an aphid by immunocytochemistry (synapsin, serotonin, and several neuropeptides), ethyl-gallate staining, confocal laser scanning microscopy, and three-dimensional reconstructions. The study has revealed well-developed optic lobes composed of lamina, medulla, and lobula complex. Ocelli are only present in males and winged parthenogenetic females. The central complex is well-defined, with a central body divided into two parts, a protocerebral bridge, and affiliated lateral accessory lobes. The mushroom bodies are ill-defined, lacking calyces, and only being visualized by using an antiserum against the neuropeptide orcokinin. The antennal lobes contain poorly delineated glomeruli but can be clearly visualized by performing antennal backfills. On the basis of our detailed description of the brain of winged and wingless parthenogenetic A. pisum females, an anatomical map is now available that should improve our knowledge of the way that these structures are involved in the regulation of phenotypic plasticity.

Ordinary glomeruli in the antennal lobe of male and female tortricid moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae) process sex pheromone and host-plant volatiles.

Both sexes of Grapholita molesta, a key pest of stone fruits, are able to detect host-plant volatiles and the sex pheromone emitted by females, and to modify their behaviour accordingly. How olfactory information is processed in the central nervous system is unknown. Intracellular recordings and stainings were used to characterize antennal lobe (AL) neuron responses to single pheromone components, a behaviourally active blend of five peach volatiles and a pear-fruit ester. AL neurons with different response patterns responded to pheromone components and plant volatiles. In males more neurons responded specifically to the main pheromone component than in females, whereas neurons responding to all three pheromone components were more abundant in females. Neurons responding to all three pheromone components often responded also to the tested plant volatiles in both sexes. Responses to all pheromone components were dose dependent in males and females, but dose-response relationships differed between neurons and tested pheromone components. Among the five AL projection neurons identified neuroanatomically in males, no arborizations were observed in the enlarged cumulus (Cu), although all of them responded to pheromone compounds. In one of two stained projection neurons in females, however, the glomerulus, which is thought to be homologous to the Cu, was targeted. The processing of pheromone information by ordinary glomeruli rather than by the macroglomerular complex is thus a striking feature of this species, indicating that pheromone and plant volatile processing are not entirely separate in this tortricid moth AL. However, the absence of recorded pheromone responses in the Cu needs to be confirmed.

Alarm Pheromone Responses Depend on Genotype, but Not on the Presence of Facultative Endosymbionts in the Pea Aphid Acyrthosiphon pisum.

Aphids use an alarm pheromone, E-ß farnesene (EBF), to warn conspecifics of potential danger. The antennal sensitivity and behavioural escape responses to EBF can be influenced by different factors. In the pea aphid, Acyrthosiphon pisum, different biotypes are adapted to different legume species, and within each biotype, different genotypes exist, which can carry or not Hamiltonella defensa, a bacterial symbiont that can confer protection against natural enemies. We investigate here the influence of the aphid genotype and symbiotic status on the escape behaviour using a four-way olfactometer and antennal sensitivity for EBF using electroantennograms (EAGs). Whereas the investigated three genotypes from two biotypes showed significantly different escape and locomotor behaviours in the presence of certain EBF doses, the infection with H. defensa did not significantly modify the escape behaviour and only marginally influenced the locomotor behaviour at high doses of EBF. Dose-response curves of EAG amplitudes after stimulation with EBF differed significantly between aphid genotypes in correlation with behavioural differences, whereas antennal sensitivity to EBF did not change significantly as a function of the symbiotic status. The protective symbiont H. defensa does thus not modify the olfactory sensitivity to the alarm pheromone. How EBF sensitivity is modified between genotypes or biotypes remains to be investigated.