Concurrent modulation of neuronal and behavioural olfactory responses to sex and host plant cues in a male moth

Mating has profound effects on animal physiology and behaviour, not only in females but also in males, which we show here for olfactory responses. In cotton leafworm moths, Spodoptera littoralis, odour-mediated attraction to sex pheromone and plant volatiles are modulated after mating, producing a behavioural response that matches the physiological condition of the male insect. Unmated males are attracted by upwind flight to sex pheromone released by calling females, as well as to volatiles of lilac flowers and green leaves of the host plant cotton, signalling adult food and mating sites, respectively. Mating temporarily abolishes male attraction to females and host plant odour, but does not diminish attraction to flowers. This behavioural modulation is correlated with a response modulation in the olfactory system, as shown by electro-physiological recordings from antennae and by functional imaging of the antennal lobe, using natural odours and synthetic compounds. An effect of mating on the olfactory responses to pheromone and cotton plant volatiles but not to lilac flowers indicates the presence of functionally independent neural circuits within the olfactory system. Our results indicate that these circuits interconnect and weigh perception of social and habitat odour signals to generate appropriate behavioural responses according to mating state.     

Differential interactions of sex pheromone and plant odour in the olfactory pathway of a male moth

Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization.     

Changes in Odor Background Affect the Locomotory Response to Pheromone in Moths

Many animals rely on chemical cues to recognize and locate a resource, and they must extract the relevant information from a complex and changing odor environment. For example, in moths, finding a mate is mediated by a sex pheromone, which is detected in a rich environment of volatile plant compounds. Here, we investigated the effects of a volatile plant background on the walking response of male Spodoptera littoralis to the female pheromone. Males were stimulated by combining pheromone with one of three plant compounds, and their walking paths were recorded with a locomotion compensator and analyzed. We found that the addition of certain volatile plant compounds disturbed the orientation toward the sex pheromone. The effect on locomotion was correlated with the capacity of the plant compound to antagonize pheromone detection by olfactory receptor neurons, suggesting a masking effect of the background over the pheromone signal. Moths were more sensitive to changes in background compared to a constant background, suggesting that a background odor also acts as a distracting stimulus. Our experiments show that the effects of odorant background on insect responses to chemical signals are complex and cannot be explained by a single mechanism.     

Glomerular interactions in olfactory processing channels of the antennal lobes

An open question in olfactory coding is the extent of interglomerular connectivity: do olfactory glomeruli and their neurons regulate the odorant responses of neurons innervating other glomeruli? In the olfactory system of the moth Manduca sexta, the response properties of different types of antennal olfactory receptor cells are known. Likewise, a subset of antennal lobe glomeruli has been functionally characterized and the olfactory tuning of their innervating neurons identified. This provides a unique opportunity to determine functional interactions between glomeruli of known input, specifically, (1) glomeruli processing plant odors and (2) glomeruli activated by antennal stimulation with pheromone components of conspecific females. Several studies describe reciprocal inhibitory effects between different types of pheromone-responsive projection neurons suggesting lateral inhibitory interactions between pheromone component-selective glomerular neural circuits. Furthermore, antennal lobe projection neurons that respond to host plant volatiles and innervate single, ordinary glomeruli are inhibited during antennal stimulation with the female’s sex pheromone. The studies demonstrate the existence of lateral inhibitory effects in response to behaviorally significant odorant stimuli and irrespective of glomerular location in the antennal lobe. Inhibitory interactions are present within and between olfactory subsystems (pheromonal and non-pheromonal subsystems), potentially to enhance contrast and strengthen odorant discrimination.     

Interactions of mechanical stimuli and sex pheromone information in antennal lobe neurons of a male moth,<Emphasis Type="Italic"> Spodoptera littoralis</Emphasis>

Male moths respond to sex pheromone sources with up-wind flight behaviour. Localization of the odour source requires not only detection of the olfactory stimulus, but also other sensory input regarding, e.g. visual and mechanical stimuli. Thus, integration of different types of sensory input is necessary. It is, however, not known where in the central nervous system the integration of information regarding different sensory modalities takes place. Using intracellular recording and staining techniques, we investigated neurons in the antennal lobe of Spodoptera littoralis, during stimulation with a mechanical stimulus and a sex pheromone. Fifteen percent of all the neurons investigated responded to the mechanical stimulus and the majority of these neurons showed altered responses if the olfactory stimulus was added. A receptor neuron responding only to the wind stimulus was found to arborise in the antennal lobe. Most projection neurons responded with an enhanced action potential frequency to the combined stimulus. In local interneurons, enhancement, depression, or no change of the responses to the wind stimulus was found when the olfactory stimulus was added. The results suggest that neurons present in the antennal lobe integrate mechanosensory and olfactory input, possibly assisting the moths to orient during up-wind flight towards an odour source.     

Increased behavioral and neuronal sensitivity to sex pheromone after brief odor experience in a moth

Plasticity in the response to stimuli related to food and oviposition cues is well documented in insects. However, responses to cues related to reproduction, for example, sex pheromones, are considered to be innate and thus not affected by experience. Here we show that brief preexposure to sex pheromones, without ensuing reward, lowers the threshold for behavioral response and augments the sensitivity in antennal lobe interneurons to pheromone compared with naive male moths. Thus, the sex pheromone system in insects can be modulated by experience. In addition, we show that the behavioral attraction to sex pheromone increases after preexposure in a time-dependent manner: a short-term effect, possibly a form of sensitization, and a long-term effect after more than 24 h. The behavioral long-term effect is paralleled by an increase in sensitivity of interneurons in the primary olfactory center, whereas the peripheral olfactory system does not change its sensitivity. We hypothesize that short-term sensitization to sex pheromone serves as a kind of alert system, whereas the long-term effect improves male performance when reproductively active females are present.     

Antennal responses of Cydia pomonella (L.) exposed to surfaces treated with methoxyfenozide

Electroantennogram (EAG) measurements were recorded from the antennae of male and female codling moth, Cydia pomonella L., to determine whether adult moths exposed to surfaces treated with the ecdysteroid agonist methoxyfenozide experience a decline in their antennal reception and thus olfactory sensitivity. Such a phenomenon would offer a possible mechanism for the previously reported decreased responsiveness from moths treated with methoxyfenozide to pheromone‐ and plant volatile‐based monitoring lures. Mean EAG data revealed that the antennae from methoxyfenozide‐treated male moths appear to be just as sensitive to various doses of synthetic codlemone as the antennae from the control and surfactant‐treated moths, but they appeared to be less sensitive to the pheromone component 12OH (collected from female effluvia) than the control male antennae. Mean male EAG responses to the pheromone components E8,E10‐12Al and codlemone collected from methoxyfenozide‐treated females were significantly less than the responses towards those two pheromone components collected from the control and surfactant‐treated females. Female moth exposure to methoxyfenozide did not negatively impact the sensitivity of female antennae to the plant volatile pear ester, but it did towards the apple volatile butyl hexanoate. Data from this study show that adult C. pomonella exposure to methoxyfenozide‐treated surfaces does appear to negatively impact, in a minor way, the (i) olfactory sensitivity (or detection) of male antennae towards some components of the female sex pheromone, (ii) the female antennal sensitivity towards a key apple volatile and (iii) the attractiveness of female pheromone effluvia.     

Plant-odour-specific receptor neurones on the antennae of female and male Spodoptera littoralis

Abstract. Receptor neurones with high selectivity and sensitivity to plant odours were found within short sensilla trichodea on the antenna of both female and male Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) by using single-sensillum recording techniques. In 112 sensilla from females and forty-one from males, twenty-four different receptor neurone types were characterized according to their specificity. Altogether, twenty-six plant and three sex pheromone compounds were tested. Receptor neurones responding with high specificity to flower odours, green leaf volatiles, oviposition deterrents and other general host plant odours were identified. In twenty-one receptor neurone types, responses were elicited by one or several plant compounds, and in three types responses were elicited by sex pheromone compounds. The majority of the receptor neurones responded to only one or two of the tested compounds. In general, only one of the two receptor neurones in a sensillum responded to any of the compounds tested. An exception was a receptor neurone responding to plant odours (green leaf volatiles) and another receptor neurone responding to a sex pheromone compound ([Z]-7-dodecenyl (acetate), which occurred in the same sensillum. The majority of the receptor neurones displayed a high sensitivity to plant odours. No morphological difference was identified the different sensillum types.     

Sex pheromone and plant-associated odour processing in antennal lobe interneurons of male Spodoptera littoralis (Lepidoptera: Noctuidae)

Antennal lobe interneurons of male Spodoptera littoralis (Boisd.) were investigated by using intracellular recording and staining techniques. Physiological and morphological characteristics of local interneurons and projection neurons responding to sex pheromone and plant-associated volatiles are described. The interneurons identified were divided into three groups, depending on their physiological response characteristics. Both types of interneurons, local interneurons and projection neurons, were described in all three groups. 1. Interneurons responding exclusively to sex pheromone stimuli, displayed different degrees of specificity. These neurons responded to either one, two, three or all four of the single sex pheromone or sex pheromone-like compounds tested. Most of these neurons also responded to a mixture of the two pheromone components present in the female S. littoralis blend. Two local interneurons and one projection neuron were identified as blend specialists, not responding to the single female produced sex pheromone components, but only to their mixture. Five pheromone specific projection neurons arborized in one or more subcompartments of the macroglomercular complex (MGC) and some of them had axonal branches in the calyces of the mushroom body and in different parts of the lateral protocerebrum. 2. Interneurons responding only to plant-associated volatiles varied highly in specificity. Neurons responding to only one of the stimuli, neurons responding to a variety of different odours and one neuron responding to all stimuli tested, were found. Three specialized local interneurons had arborizations only in ordinary glomeruli. One specialized and three less specialized local interneurons had arborizations within the MGC and the ordinary glomeruli. The projection neurons responding only to plant-associated volatiles had mostly uni- or multiglomerular arborizations within the ordinary glomeruli. 3. Interneurons responding to both sex pheromones and plant-associated stimuli varied in specificity. Individual interneurons that responded to few plant-associated odours mostly responded to several pheromone stimuli as well. Projection neurons responding to most of the plant-associated volatiles also responded to all pheromone stimuli. Two local interneurons responding to both stimulus groups, arborized within the MGC and the ordinary glomeruli. Projection neurons mostly arborized in only one ordinary glomerulus or in one compartment of the MGC. The variation in specificity and sensitivity of antennal lobe interneurons and structure-function correlations are discussed.     

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.     

Olfactory receptor neurons detecting plant odours and male volatiles in Anomala cuprea beetles (Coleoptera: Scarabaeidae)

We have identified several types of olfactory receptor neurons in male and female Anomala cuprea beetles. The receptor neurons were sensitive to female sex pheromone components, flower volatiles, green leaf volatiles and unknown volatiles from males. Olfactory sensilla were located on three lamellae forming the antennal club. There was a clear spatial separation between some types of sensilla on each lamella. Receptor neurons for the two sex pheromone components were situated in sensilla placodea covering a specific area on each lamella in both males and females. All sex pheromone receptor neurons were found in these sensilla. Most other receptor neurons were located in a longitudinal, heterogeneous streak formed by various types of sensilla. Receptor neurons for plant-derived compounds appeared to be specialists with a high sensitivity to their respective key compound. The most remarkable among these are the green leaf volatile-specific receptor neurons, which were both sensitive and selective, with the key compound being at least 1000 times as effective as any other compound. These green leaf volatile detectors are apparently homologous to detectors recently found in the scarab Phyllopertha diversa. Our results emphasize the role of single-sensillum recordings as a tool in the identification of biologically active odours.     

Olfactory activation patterns in the antennal lobe of the sphinx moth,<Emphasis Type="Italic"> Manduca sexta</Emphasis>

The sphinx moth Manduca sexta is a well-studied insect with regard to central olfactory functions. Until now, the innervation patterns of olfactory receptor neurons into the array of olfactory glomeruli in the antennal lobe have, however, been unclear. Using optical imaging to visualize calcium dynamics within the antennal lobe we demonstrate specific patterns elicited by sex pheromone components and plant-derived odours. These patterns mainly reflect receptor neuron activity. Within the male-specific macroglomerular complex the two major pheromone components evoke stereotyped activity in either of two macroglomerular complex glomeruli. Based on previous knowledge of output neuron specificity, our results suggest a matching of information between input and output in the macroglomerular complex. Plant odours evoked activity in the sexually isomorphic glomeruli. Two major results were obtained: (1). terpenes and aromatic compounds activate different clusters of glomeruli with only minor overlapping, and (2). the position of certain key glomeruli is fixed in both males and females, which suggests that host-plant related odorants are processed in a similar way in both sexes.     

Calcium responses to pheromones and plant odours in the antennal lobe of the male and female moth Heliothis virescens

In male moths, the primary olfactory integration centre, the antennal lobe, consists of two systems. The macroglomerular complex processes pheromone information, while the ordinary glomeruli process plant odour information. Females lack a macroglomerular complex. We measured the spatial representation of odours using in-vivo optical recording. We found that: (1) pheromone substances elicited activity exclusively in the MGC. No response was found in female antennal lobes. (2) Plant odours elicited combinatorial activity patterns in the ordinary glomeruli in both males and females. No response was found in the MGC of male moths. (3) A clean air puff often led to activity, in both males and females, suggesting that mechano-sensory information is also processed in the antennal lobe. (4) With an interstimulus interval of 5 or 10 s, strongly activated glomeruli were able to follow the temporal structure of the stimulus, while others lost their phase-locking. Some glomeruli showed "off" responses. These properties were odour dependent. This confirms and extends previous studies, showing the functional significance of the two subsystems for processing olfactory information. Pheromones are coded in a combinatorial manner within the macroglomerular complex, with each glomerulus corresponding to one information channel. Plant odours are coded in an across-glomeruli code in the ordinary glomeruli.     

Representation of primary plant odorants in the antennal lobe of the moth Heliothis virescens using calcium imaging

The primary olfactory centre, the antennal lobe of Heliothis virescens moths, contains 62 glomeruli which process plant odour information and four male-specific glomeruli which form the macroglomerular complex, involved in processing information about pheromone and interspecific signals. Using calcium imaging, we recorded the spatio-temporal activity pattern of the glomeruli in the anterior antennal lobe during stimulation with odorants produced by plants or insects. Each odorant elicited specific excitatory responses in one or a few glomeruli: the major pheromone component did so exclusively in the large glomerulus of the macroglomerular complex and the plant odours exclusively in the ordinary glomeruli. Eight glomeruli, with corresponding plant odour responses and positions, were identified within each sex. Glomeruli responded specifically to linalool, beta-ocimene/beta-myrcene or germacrene D/alpha-farnesene. Responses to two essential plant oils covered the response areas of their major constituents, as well as activating additional glomeruli. Stronger activation in the AL due to increased odour concentration was expressed as increased response strength within the odorant-specific glomeruli as well as recruitment of less sensitive glomeruli.     

Sex pheromone of the cotton leafworm, Spodoptera littoralis

cis-9,trans-11-Tetradecadien-1-ol acetate (A) and cis-9,trans-12-tetradecadien-1-ol acetate (B), the same compounds as the female sex pheromone of Spodoptera litura, were identified as major components of the female sex pheromone of the cotton leafworm, Spodoptera littoralis. Compounds A and B were individually active, but the activity was synergistically enhanced by mixing the two compounds. The male response was optimal with mixture ranging from 5 : 1 to 20 : 1 of compounds A and B, respectively. Fifty per cent of the male moths of S. littoralis responded to the 20 : 1 mixture at the 10^?7 μg level; they responded at a concentration 100-fold lower than did male moths of S. litura. A partially purified extract of females of S. litura stimulated male moths of S. littoralis. Similarly a partially purified extract of S. littoralis was also active for the male moths of S. litura. The extract of females of S. litura, however, was 1000 times less active for male moths of S. littoralis than was the exrtact of females of S. littoralis, suggesting that a compound(s) is present in the extract of S. litura that inhibits the response of males of S. littoralis.     

Structure and function of the antennal sensilla of the palm weevil Rhynchophorus palmarum (Coleoptera, Curculionidae)

The distribution, fine structure and function of the sensilla present on the antennal club of Rhynchophorus palmarum were studied. No sex dimorphism was observed. Scanning and transmission electron microscopy showed five types of hair-like structures, four of which were evenly distributed on the antennal club. Two types of hair (IV and V) showed wall pores, a characteristic of olfactory sensilla. The antenna numbers 11,190 +/- 3040 type IV and 7360 +/- 1500 type V hairs. Using single sensillum recording, we identified 17 types of olfactory receptor neurons (ORNs) on the basis of their responses to pheromone and host plant odors, triggering synergic behavioral responses. We characterized highly specific and sensitive ORNs tuned to the aggregation pheromone (18% ORNs; 0.01-1 ng response threshold) and to host plant odors such as ethyl acetate, ethanol, acetoin and guaiacol (10% ORNs; 1-10 ng response threshold). Eleven percent of the ORNs were more generalist, responding to several odors with low sensitivity. Nine percent of the ORNs showed a complex pattern of responses, being co-activated by the pheromone and plant odors. This suggests an interaction at the sensory neuron level between pheromone and plant odors, triggering synergic behavioral responses.     

Modelling efficiency in insect olfactory information processing

The olfactory system of insects is essential for the search of food and mates, and weak signals can be detected, amplified and discriminated in a fluctuating environment. The olfactory system also allows for learning and recall of odour memories. Based on anatomical, physiological, and behavioural data from the olfactory system of insects, we have developed a cross-scale dynamical neural network model to simulate the presentation, amplification and discrimination of host plant odours and sex pheromones. In particular, we model how the spatial and temporal patterns of the odour information emerging in the glomeruli of the antennal lobe (AL) rely on the glomerular morphology, the connectivity and the complex dynamics of the AL circuits. We study how weak signals can be amplified, how different odours can be discriminated, based on stochastic (resonance) dynamics and the connectivity of the network. We further investigate the spatial and temporal coding of sex pheromone components and plant volatile compounds, in relation to the glomerular structure, arborizing patterns of the projection neurons (PNs) and timing patterns of the neuronal spiking activity.