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.

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.

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.

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.

Sublethal Exposure Effects of the Neonicotinoid Clothianidin Strongly Modify the Brain Transcriptome and Proteome in the Male Moth Agrotis ipsilon.

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids such as clothianidin. The residual accumulation of low concentrations of these insecticides can have positive effects on target 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 indeed modify chemical communication. We recently showed that treatments with low doses of clothianidin could induce hormetic effects on behavioral and neuronal sex pheromone responses in the male moth, Agrotis ipsilon. In this study, we used high-throughput RNAseq and proteomic analyses from brains of A. ipsilon males that were intoxicated with a low dose of clothianidin to investigate the molecular mechanisms leading to the observed hormetic effect. Our results showed that clothianidin induced significant changes in transcript levels and protein quantity in the brain of treated moths: 1229 genes and 49 proteins were differentially expressed upon clothianidin exposure. In particular, our analyses highlighted a regulation in numerous enzymes as a possible detoxification response to the insecticide and also numerous changes in neuronal processes, which could act as a form of acclimatization to the insecticide-contaminated environment, both leading to enhanced neuronal and behavioral responses to sex pheromone.

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.

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.

The Antennal Pathway of Dragonfly Nymphs, from Sensilla to the Brain.

Dragonflies are hemimetabolous insects, switching from an aquatic life style as nymphs to aerial life as adults, confronted to different environmental cues. How sensory structures on the antennae and the brain regions processing the incoming information are adapted to the reception of fundamentally different sensory cues has not been investigated in hemimetabolous insects. Here we describe the antennal sensilla, the general brain structure, and the antennal sensory pathways in the last six nymphal instars of Libellula depressa, in comparison with earlier published data from adults, using scanning electron microscopy, and antennal receptor neuron and antennal lobe output neuron mass-tracing with tetramethylrhodamin. Brain structure was visualized with an anti-synapsin antibody. Differently from adults, the nymphal antennal flagellum harbors many mechanoreceptive sensilla, one olfactory, and two thermo-hygroreceptive sensilla at all investigated instars. The nymphal brain is very similar to the adult brain throughout development, despite the considerable differences in antennal sensilla and habitat. Like in adults, nymphal brains contain mushroom bodies lacking calyces and small aglomerular antennal lobes. Antennal fibers innervate the antennal lobe similar to adult brains and the gnathal ganglion more prominently than in adults. Similar brain structures are thus used in L. depressa nymphs and adults to process diverging sensory information.

Insect olfactory communication in a complex and changing world.

Insect intraspecific olfactory communication occurs in a complex sensory environment. Here we present recent results on how the olfactory system extracts specific information from a sensory background, and integrates it with complementary information to improve odor source localization. Recent advances on mechanisms of olfactory mixture processing, multi-modal integration, as well as plasticity of sensory processing are reviewed. Significant progress in the understanding of neural coding and molecular bases of olfaction reinforce our perception of the tremendous adaptability of insects to a changing environment. However several reports demonstrate that anthropogenic environmental perturbations interfere with insect olfactory communication and might as a consequence significantly alter the functioning of ecosystems and agroecosystems.

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.

Neuroanatomical correlates of mobility: Sensory brain centres are bigger in winged than in wingless parthenogenetic pea aphid females.

Many aphid species reproduce parthenogenetically throughout most of the year, with individuals having identical genomes. Nevertheless, aphid clones display a marked polyphenism with associated behavioural differences. Pea aphids (Acyrthosiphon pisum), when crowded, produce winged individuals, which have a larger dispersal range than wingless individuals. We examined here if brain structures linked to primary sensory processing and high-order motor control change in size as a function of wing polyphenism. Using micro-computing tomography (micro-CT) scans and immunocytochemical staining with anti-synapsin antibody, we reconstructed primary visual (optic lobes) and olfactory (antennal lobes) neuropils, together with the central body of winged and wingless parthenogenetic females of A. pisum for volume measurements. Absolute neuropil volumes were generally bigger in anti-synapsin labelled brains compared to micro-CT scans. This is potentially due to differences in rearing conditions of the used aphids. Independent of the method used, however, winged females consistently had larger antennal lobes and optic lobes than wingless females in spite of a larger overall body size of wingless compared to winged females. The volume of the central body, on the other hand was not significantly different between the two morphs. The larger primary sensory centres in winged aphids might thus provide the neuronal substrate for processing different environmental information due to the increased mobility during flight.

Early Olfactory Environment Influences Antennal Sensitivity and Choice of the Host-Plant Complex in a Parasitoid Wasp.

Early experience of olfactory stimuli associated with their host-plant complex (HPC) is an important driver of parasitoid foraging choices, notably leading to host fidelity. Mechanisms involved, such as peripheral or central modulation, and the impact of a complex olfactory environment are unknown. Using olfactometer assays, we compared HPC preference of Aphidius ervi Haliday (Hymenoptera:Braconidae) females originating from two different HPCs, either with the other HPC in close vicinity (complex environment) or without (simple environment). We also investigated antennal responses to volatiles differentially emitted by the two respective HPCs. In a simple environment, HPC of origin had an influence on olfactory choice, but the preferences observed were asymmetric according to parasitoid origin. Electroantennographic recordings revealed significant sensitivity differences for some of the tested individual volatiles, which are emitted differentially by the two HPCs. Besides, presence of an alternative HPC during early stages modified subsequent parasitoid preferences. We discuss how increased olfactory complexity could influence parasitoid host foraging and biological control in diversified cropping systems.

Acibenzolar-S-Methyl Reprograms Apple Transcriptome Toward Resistance to Rosy Apple Aphid.

Acibenzolar-S-methyl (ASM) is a chemical compound, which is able to induce resistance in several model and non-model plants, but the end-players of this induced defense remain ill-defined. Here, we test the hypothesis that treatment with ASM can protect apple (Malus × domestica) against the rosy apple aphid (Dysaphis plantaginea) and investigate the defense molecules potentially involved in resistance. We measured aphid life traits and performed behavioral assays to study the effect of ASM on plant resistance against the aphid, and then combined transcriptomic, bioinformatics, metabolic and biochemical analyses to identify the plant compounds involved in resistance. Plants treated with ASM negatively affected several life traits of the aphid and modified its feeding and host seeking behaviors. ASM treatment elicited up-regulation of terpene synthase genes in apple and led to the emission of (E,E)-α-farnesene, a sesquiterpene that was repellent to the aphid. Several genes encoding amaranthin-like lectins were also strongly up-regulated upon treatment and the corresponding proteins accumulated in leaves, petioles and stems. Our results link the production of specific apple proteins and metabolites to the antibiosis and antixenosis effects observed against Dysaphis plantaginea, providing insight into the mechanisms underlying ASM-induced herbivore resistance.

A Background of a Volatile Plant Compound Alters Neural and Behavioral Responses to the Sex Pheromone Blend in a Moth.

Recognition of intra-specific olfactory signals within a complex environment of plant-related volatiles is crucial for reproduction in male moths. Sex pheromone information is detected by specific olfactory receptor neurons (Phe-ORNs), highly abundant on the male antenna. The information is then transmitted to the pheromone processing macroglomerular complex (MGC) within the primary olfactory center, the antennal lobe, where it is processed by local interneurons and projection neurons. Ultimately a behavioral response, orientation toward the pheromone source, is elicited. Volatile plant compounds (VPCs) are detected by other functional types of olfactory receptor neurons (ORNs) projecting in another area of the antennal lobe. However, Phe-ORNs also respond to some VPCs. Female-produced sex pheromones are emitted within a rich environment of VPCs, some of which have been shown to interfere with the detection and processing of sex pheromone information. As interference between the different odor sources might depend on the spatial and temporal features of the two types of stimuli, we investigated here behavioral and neuronal responses to a brief sex pheromone blend pulse in a VPC background as compared to a control background in the male noctuid moth Agrotis ipsilon. We observed male orientation behavior in a wind tunnel and recorded responses of Phe-ORNs and MGC neurons to a brief sex pheromone pulse within a background of individual VPCs. We also recorded the global input signal to the MGC using in vivo calcium imaging with the same stimulation protocol. We found that VPCs eliciting a response in Phe-ORNs and MGC neurons masked responses to the pheromone and decreased the contrast between background odor and the sex pheromone at both levels, whereas α-pinene did not interfere with first order processing. The calcium signal produced in response to a VPC background was tonic, lasting longer than the VPC stimulus duration, and masked entirely the pheromone response. One percent heptanal and linalool, in addition to the masking effect, caused a clear delay in responses of MGC neurons to the sex pheromone. Upwind flight toward the pheromone in a wind tunnel was also delayed but otherwise not altered by different doses of heptanal.

An Insecticide Further Enhances Experience-Dependent Increased Behavioural Responses to Sex Pheromone in a Pest Insect.

Neonicotinoid insecticides are widely used to protect plants against pest insects, and insecticide residues remaining in the environment affect both target and non-target organisms. Whereas low doses of neonicotinoids have been shown to disturb the behaviour of pollinating insects, recent studies have revealed that a low dose of the neonicotinoid clothianidin can improve behavioural and neuronal sex pheromone responses in a pest insect, the male moth Agrotis ipsilon, and thus potentially improve reproduction. As male moth behaviour depends also on its physiological state and previous experience with sensory signals, we wondered if insecticide effects would be dependent on plasticity of olfactory-guided behaviour. We investigated, using wind tunnel experiments, whether a brief pre-exposure to the sex pheromone could enhance the behavioural response to this important signal in the moth A. ipsilon at different ages (sexually immature and mature males) and after different delays (2 h and 24 h), and if the insecticide clothianidin would interfere with age effects or the potential pre-exposure-effects. Brief pre-exposure to the pheromone induced an age-independent significant increase of sex pheromone responses 24 h later, whereas sex pheromone responses did not increase significantly 2 h after exposure. However, response delays were significantly shorter compared to naïve males already two hours after exposure. Oral treatment with clothianidin increased sex pheromone responses in sexually mature males, confirming previous results, but did not influence responses in young immature males. Males treated with clothianidin after pre-exposure at day 4 responded significantly more to the sex pheromone at day 5 than males treated with clothianidin only and than males pre-exposed only, revealing an additive effect of experience and the insecticide. Plasticity of sensory systems has thus to be taken into account when investigating the effects of sublethal doses of insecticides on behaviour.