Volatile-mediated oviposition preference for healthy over root-infested plants by the European corn borer.

The selection of oviposition sites by female moths is crucial in shaping their progeny performance and survival, and consequently in determining insect fitness. Selecting suitable plants that promote the performance of the progeny is referred to as the Preference-Performance hypothesis (or 'mother-knows-best'). While root infestation generally reduces the performance of leaf herbivores, little is known about its impact on female oviposition. We investigated whether maize root infestation by the Western corn rootworm (WCR) affects the oviposition preference and larval performance of the European corn borer (ECB). ECB females used leaf volatiles to select healthy plants over WCR-infested plants. Undecane, a compound absent from the volatile bouquet of healthy plants, was the sole compound to be upregulated upon root infestation and acted as a repellent for first oviposition. ECB larvae yet performed better on plants infested below-ground than on healthy plants, suggesting an example of 'bad motherhood'. The increased ECB performance on WCR-infested plants was mirrored by an increased leaf consumption, and no changes in the plant primary or secondary metabolism were detected. Understanding plant-mediated interactions between above- and below-ground herbivores may help to predict oviposition decisions, and ultimately, to manage pest outbreaks in the field.

Seasonal changes in photoperiod and temperature lead to changes in cuticular hydrocarbon profiles and affect mating success in Drosophila suzukii.

Seasonal plasticity in insects is often triggered by temperature and photoperiod changes. When climatic conditions become sub-optimal, insects might undergo reproductive diapause, a form of seasonal plasticity delaying the development of reproductive organs and activities. During the reproductive diapause, the cuticular hydrocarbon (CHC) profile, which covers the insect body surface, might also change to protect insects from desiccation and cold temperature. However, CHCs are often important cues and signals for mate recognition and changes in CHC composition might affect mate recognition. In the present study, we investigated the CHC profile composition and the mating success of Drosophila suzukii in 1- and 5-day-old males and females of summer and winter morphs. CHC compositions differed with age and morphs. However, no significant differences were found between the sexes of the same age and morph. The results of the behavioral assays show that summer morph pairs start to mate earlier in their life, have a shorter mating duration, and have more offspring compared to winter morph pairs. We hypothesize that CHC profiles of winter morphs are adapted to survive winter conditions, potentially at the cost of reduced mate recognition cues.

Elevated ozone and carbon dioxide affects the composition of volatile organic compounds emitted by Vicia faba (L.) and visitation by European orchard bee (Osmia cornuta).

Recent studies link increased ozone (O3) and carbon dioxide (CO2) levels to alteration of plant performance and plant-herbivore interactions, but their interactive effects on plant-pollinator interactions are little understood. Extra floral nectaries (EFNs) are essential organs used by some plants for stimulating defense against herbivory and for the attraction of insect pollinators, e.g., bees. The factors driving the interactions between bees and plants regarding the visitation of bees to EFNs are poorly understood, especially in the face of global change driven by greenhouse gases. Here, we experimentally tested whether elevated levels of O3 and CO2 individually and interactively alter the emission of Volatile Organic Compound (VOC) profiles in the field bean plant (Vicia faba, L., Fabaceae), EFN nectar production and EFN visitation by the European orchard bee (Osmia cornuta, Latreille, Megachilidae). Our results showed that O3 alone had significant negative effects on the blends of VOCs emitted while the treatment with elevated CO2 alone did not differ from the control. Furthermore, as with O3 alone, the mixture of O3 and CO2 also had a significant difference in the VOCs' profile. O3 exposure was also linked to reduced nectar volume and had a negative impact on EFN visitation by bees. Increased CO2 level, on the other hand, had a positive impact on bee visits. Our results add to the knowledge of the interactive effects of O3 and CO2 on plant volatiles emitted by Vicia faba and bee responses. As greenhouse gas levels continue to rise globally, it is important to take these findings into consideration to better prepare for changes in plant-insect interactions.

The Comparative Toxicity, Biochemical and Physiological Impacts of Chlorantraniliprole and Indoxacarb on Mamestra brassicae (Lepidoptera: Noctuidae).

BACKGROUND: The cabbage moth, Mamestra brassicae, is a polyphagous pest that attacks several crops. Here, the sublethal and lethal effects of chlorantraniliprole and indoxacarb were investigated on the developmental stages, detoxification enzymes, reproductive activity, calling behavior, peripheral physiology, and pheromone titer of M. brasssicae. Methods: To assess pesticide effects, the second instar larvae were maintained for 24 h on a semi-artificial diet containing insecticides at their LC10, LC30, and LC50 concentrations. RESULTS: M. brassicae was more susceptible to chlorantraniliprole (LC50 = 0.35 mg/L) than indoxacarb (LC50 = 1.71 mg/L). A significantly increased developmental time was observed with both insecticides at all tested concentrations but decreases in pupation rate, pupal weight, and emergence were limited to the LC50 concentration. Reductions in both the total number of eggs laid per female and the egg viability were observed with both insecticides at their LC30 and LC50 concentrations. Both female calling activity and the sex pheromone (Z11-hexadecenyl acetate and hexadecenyl acetate) titer were significantly reduced by chlorantraniliprole in LC50 concentration. Antennal responses of female antennae to benzaldehyde and 3-octanone were significantly weaker than controls after exposure to the indoxocarb LC50 concentration. Significant reductions in the enzymatic activity of glutathione S-transferases, mixed-function oxidases, and carboxylesterases were observed in response to both insecticides.

The Alarm Pheromone and Alarm Response of the Clonal Raider Ant.

Ants communicate via an arsenal of different pheromones produced in a variety of exocrine glands. For example, ants release alarm pheromones in response to danger to alert their nestmates and to trigger behavioral alarm responses. Here we characterize the alarm pheromone and the alarm response of the clonal raider ant Ooceraea biroi, a species that is amenable to laboratory studies but for which no pheromones have been identified. During an alarm response, ants quickly become unsettled, leave their nest pile, and are sometimes initially attracted to the source of alarm, but ultimately move away from it. We find that the alarm pheromone is released from the head of the ant and identify the putative alarm pheromone as a blend of two compounds found in the head, 4-methyl-3-heptanone and 4-methyl-3-heptanol. These compounds are sufficient to induce alarm behavior alone and in combination. They elicit similar, though slightly different behavioral features of the alarm response, with 4-methyl-3-heptanone being immediately repulsive and 4-methyl-3-heptanol being initially attractive before causing ants to move away. The behavioral response to these compounds in combination is dose-dependent, with ants becoming unsettled and attracted to the source of alarm pheromone at low concentrations and repulsed at high concentrations. While 4-methyl-3-heptanone and 4-methyl-3-heptanol are known alarm pheromones in other more distantly related ant species, this is the first report of the chemical identity of a pheromone in O. biroi, and the first alarm pheromone identified in the genus Ooceraea. Identification of a pheromone that triggers a robust, consistent, and conserved behavior, like the alarm pheromone, provides an avenue to dissect the behavioral and neuronal mechanisms underpinning chemical communication.

Essential Oil Headspace Volatiles Prevent Invasive Box Tree Moth (Cydalima perspectalis) Oviposition-Insights from Electrophysiology and Behaviour.

The box tree moth (Cydalima perspectalis Walker) is an invasive species in Europe causing severe damage both in natural and ornamental boxwood (Buxus spp.) vegetation. Pest management tactics are often based on the use of chemical insecticides, whereas environmentally-friendly control solutions are not available against this insect. The application of essential oils may provide effective protection against oviposition and subsequent larval damage. Oviposition deterrence of cinnamon, eucalyptus and lavender essential oils was tested on female C. perspectalis in behavioural bioassays. Our results indicate that all the studied essential oils may be adequate deterrents; however, cinnamon oil exhibited the strongest effect. To determine the physiologically active compounds in the headspace of the essential oils, gas chromatography coupled with electroantennography recordings were performed in parallel with gas chromatography-mass spectrometry to identify the volatile constituents. In addition, the release rates of various components from vial-wick dispensers were measured during the oviposition bioassay. These results may serve as a basis for the development of a practical and insecticide-free plant protection method against this invasive moth species.

Development of a Female-Targeted Lure for the Box Tree Moth Cydalima perspectalis (Lepidoptera: Crambidae): a Preliminary Report.

The box tree moth, Cydalima perspectalis, is an invasive pest in Europe causing damage on Buxus species. In this study, we aimed to develop a "bisexual" lure to attract both female and male moths. Based on a previous screening bioassay we tested methyl salicylate, phenylacetaldehyde and eugenol as potential attractants in different combinations. The trapping results showed that both binary and ternary blends attracted male and female moths. Catches with these blends were comparable to catches with the synthetic pheromone. Subsequently we carried out single sensillum recordings, which proved the peripheral detection of the above-mentioned compounds on male and female antennae. To identify synergistic flower volatiles, which can be also attractive and can increase the trap capture, we collected flower headspace volatiles from 12 different flowering plant species. Several components of the floral scents evoked good responses from antennae of both females and males in gas chromatography-electroantennographic detection. The most active components were tentatively identified by gas chromatography coupled mass spectrometry as benzaldehyde, cis-ß-ocimene, (±)-linalool and phenethyl alcohol. These selected compounds in combination did not increase significantly the trap capture compared to the methyl salicylate- phenyacetaldehyde blend. Based on these results we discovered the first attractive blend, which was able to attract both adult male and female C. perspectalis in field conditions. These results will yield a good basis for the optimization and development of a practically usable bisexual lure against this invasive pest.

False positives from impurities result in incorrect functional characterization of receptors in chemosensory studies.

The discovery of chemoreceptors and technological advances have greatly increased our understanding of chemosensory mechanisms. However, some of this rapid progress may have been severely compromised by insufficient attention given to the possible effects of impurities in the chemical standards used in identifying ligands for target receptors. Here, we show that even trace amounts of impurities in test stimuli can completely obscure true ligand-receptor relationships. Responses to impurities may go unrecognized because of two main factors. First, the sensitivity of receptors to ligands may be greater than that of the instruments used to check sample purity. Second, the concentrations of impurities actually reaching the chemoreceptor during experiments may be orders of magnitude higher than that of the putative stimulus, due to large differences in vapour pressure between the impurities and the putative stimulus. Errors caused by impurities are not limited to receptor-ligand studies, but can also affect related areas of chemosensory research, such as neural processing, downstream behaviours, and "in-silico" bioinformatics predictions of response profiles. The purity of standards is always implied but must be checked rigorously to prevent skewed or invalid results or conclusions, such as we exemplify here for Drosophila melanogaster and its olfactory receptor DmOr7a.

Benzaldehyde: an alfalfa-related compound for the spring attraction of the pest weevil Sitona humeralis (Coleoptera: Curculionidae).

BACKGROUND: Sitona weevils (Coleoptera: Curculionidae) are a species complex comprising pests of many leguminous crops worldwide, causing damage to young plants as adults and to rootlets as larvae, resulting in significant yield losses. Timely detection of migrating adult weevils is needed to determine when deployment of control measures becomes necessary. With the aim of developing plant volatile-based lures for Sitona spp. detection, we investigated the responses of S. humeralis to host plant-related aromatic compounds. RESULTS: In olfactometer studies, both male and female S. humeralis responded positively to the odour of alfalfa flowers, a source of aromatic volatiles. In single sensillum recordings, basiconic sensilla located on the third and fourth terminal segments of the antennal club of both sexes were found to respond to benzaldehyde at doses of 10-5 and 10-4  g, suggesting that the weevil is able to detect this compound at the peripheral sensory level. In field studies, S. humeralis was attracted to benzaldehyde in the spring, but not in the autumn. CONCLUSION: Benzaldehyde, as described in this study, may be a suitable candidate for the development of monitoring tools for S. humeralis. © 2019 Society of Chemical Industry.

Identification of the Female-Produced Sex Pheromone of an Invasive Greenhouse Pest, the European Pepper Moth (Duponchelia fovealis).

The European pepper moth (Duponchelia fovealis, Lepidoptera, Crambidae, Spilomelinae) is an invasive pest of greenhouses in many countries, causing serious damages to horticultural plants. Coupled gas chromatographic-electroantennographic detection analysis of the female gland extract revealed two antennally active peaks. Using coupled gas chromatography-mass spectrometry (GC-MS), one was identified as (Z)-11-hexadecenal (Z11-16:Ald); however, further analysis on different types of capillary columns indicated that the second active compound has two different isomers, (E)-13-octadecenal (E13-18:Ald) and (Z)-13-octadecenal (Z13-18:Ald). The approximate ratio of E13-18:Ald, Z13-18:Ald and Z11-16:Ald in the crude pheromone gland extract was 10:1:0.1, respectively. Single sensillum recordings showed that there was one sensory neuron that responded with a high amplitude spike to both E13-18:Ald and Z13-18:Ald, while another neuron housed in the same sensillum responded to Z11-16:Ald. Field evaluation of the identified compounds indicated that the E13-18:Ald was necessary to evoke the attraction of males; although the presence of Z13-18:Ald and Z11-16:Ald increased the catches in traps. The highest number of caught males was achieved when E13-18:Ald, Z13-18:Ald and Z11-16:Ald were present in baits in the same ratio as in the female gland extract. This pheromone can be used in a monitoring strategy and could potentially lead to the development of mating disruption.

Precision-Cut Kidney Slices as a Tool to Understand the Dynamics of Extracellular Matrix Remodeling in Renal Fibrosis.

The aim of this study was to set up an ex vivo model for renal interstitial fibrosis in order to investigate the extracellular matrix (ECM) turnover profile in the fibrotic kidney. We induced kidney fibrosis in fourteen 12-week-old male Sprague Dawley rats by unilateral ureteral obstruction (UUO) surgery of the right ureter. The left kidney (contralateral) was used as internal control. Six rats were sham operated and used as the control group. Rats were terminated two weeks after the surgery; the kidneys were excised and precision-cut kidney slices (PCKSs) were cultured for five days in serum-free medium. Markers of collagen type I formation (P1NP), collagen type I and III degradation (C1M and C3M), and α-smooth muscle actin (αSMA) were measured in the PCKS supernatants by enzyme-linked immunosorbent assay. P1NP, C1M, C3M, and α-SMA were increased up to 2- to 13-fold in supernatants of tissue slices from the UUO-ligated kidneys compared with the contralateral kidneys (P < 0.001) and with the kidneys of sham-operated animals (P < 0.0001). The markers could also reflect the level of fibrosis in different animals. The UUO PCKS ex vivo model provides a valuable translational tool for investigating the extracellular matrix remodeling associated with renal interstitial fibrosis.

Electrophysiologically-Active Maize Volatiles Attract Gravid Female European Corn Borer, Ostrinia nubilalis.

The European corn borer (ECB) is an important pest of maize in the northern hemisphere, but no reliable techniques exist for monitoring females during their reproductive period. In this study, we aimed to identify host-plant volatiles used by gravid Z-strain females in search for oviposition sites. Headspace of maize plants, to which gravid females orientated in a wind tunnel, was collected, and physiologically-active components were identified by using gas chromatography (GC) coupled with electroantennographic detection followed by GC-mass spectrometry. The antennae of female moths consistently responded to two maize volatiles, nonanal and decanal. Although these compounds are individually not characteristic for maize, a synthetic mix in a ratio found in maize headspace, 1:2.4 at 1 µg µl(-1) induced source contact and landing responses similar to maize plants in the wind tunnel. However, fewer females took flight in response to the mix, and those that took flight did so with an increased latency. To our knowledge, this is the first blend of host-plant volatiles that has been found to be physiologically active and to be able to induce attraction of gravid ECB females under laboratory conditions. Future tests will evaluate the attractiveness of the blend to the E-strain of ECB, the attractiveness of the blend in the field, and its potential in monitoring ECB populations.

Temporal features of spike trains in the moth antennal lobe revealed by a comparative time-frequency analysis.

The discrimination of complex sensory stimuli in a noisy environment is an immense computational task. Sensory systems often encode stimulus features in a spatiotemporal fashion through the complex firing patterns of individual neurons. To identify these temporal features, we have developed an analysis that allows the comparison of statistically significant features of spike trains localized over multiple scales of time-frequency resolution. Our approach provides an original way to utilize the discrete wavelet transform to process instantaneous rate functions derived from spike trains, and select relevant wavelet coefficients through statistical analysis. Our method uncovered localized features within olfactory projection neuron (PN) responses in the moth antennal lobe coding for the presence of an odor mixture and the concentration of single component odorants, but not for compound identities. We found that odor mixtures evoked earlier responses in biphasic response type PNs compared to single components, which led to differences in the instantaneous firing rate functions with their signal power spread across multiple frequency bands (ranging from 0 to 45.71 Hz) during a time window immediately preceding behavioral response latencies observed in insects. Odor concentrations were coded in excited response type PNs both in low frequency band differences (2.86 to 5.71 Hz) during the stimulus and in the odor trace after stimulus offset in low (0 to 2.86 Hz) and high (22.86 to 45.71 Hz) frequency bands. These high frequency differences in both types of PNs could have particular relevance for recruiting cellular activity in higher brain centers such as mushroom body Kenyon cells. In contrast, neurons in the specialized pheromone-responsive area of the moth antennal lobe exhibited few stimulus-dependent differences in temporal response features. These results provide interesting insights on early insect olfactory processing and introduce a novel comparative approach for spike train analysis applicable to a variety of neuronal data sets.

Intraspecific combinations of flower and leaf volatiles act together in attracting hawkmoth pollinators.

Insects pinpoint mates, food and oviposition sites by olfactory cues. Recognizing and localizing a suitable target by olfaction is demanding. Odor sources emit characteristic blends of compounds that have to be identified against an environmentally derived olfactory background. This background, however, does not necessarily disturb the localization of a source. Rather, the contrary. Sex pheromones become more attractive to male moths when being presented against a relevant plant background. Here we asked whether such olfactory coaction also characterizes foraging cues. The tobacco hornworm Manduca sexta feeds on nectar from wild tobacco Nicotiana attenuata and sacred datura Datura wrightii flowers. We tested how leaf-derived volatile blends as a background affect the moths' approach to flower blends. We found coaction when a flower blend was presented against a conspecific leaf volatile background but not when the blend was presented against volatiles emitted by the other host plant or by a non-host plant. Hence, our results reveal a species-specific coaction between flower blend and leaf volatile background. The ability to integrate information from different odor sources on one plant might provide the moth with a fine-grained analysis of food site quality.

Early quality assessment lessens pheromone specificity in a moth.

Pheromone orientation in moths is an exemplar of olfactory acuity. To avoid heterospecific mating, males respond to female-produced blends with high specificity and temporal resolution. A finely tuned sensory to projection neuron network secures specificity, and this network is thought to assess pheromone quality continually during orientation. We tested whether male moths do indeed evaluate each pheromone encounter and surprisingly found that male European corn borer moths instead generalize across successive encounters. Although initially highly ratio specific, once "locked on" to the pheromone plume the acceptable ratio can vary widely, and even unattractive blends can become attractive. We further found that this "mental shortcut" may be a consequence of the fact that sensory neurons exposed to frequent encounters do not reliably encode blend ratios. Neurons tuned to either of the two pheromone components adapt differentially in plumes containing the preferred blend ratio (97:3) and cause the olfactory sensory signal to "evolve," even in narrowly tuned pheromonal circuits. However, apparently the brain interprets these shifting signals as invariant "gestalts." Generalization in pheromone perception may mitigate stabilizing selection and allow introgression between sympatric strains, such as in the European corn borer, that otherwise appear isolated by pheromonal differences. Generalization may also be important in responses to general odorants, as circuits underlying these display vast sensitivity differences, complex interactions, and temporal intricacies.

Antennal lobe processing correlates to moth olfactory behavior.

Animals typically perceive their olfactory environment as a complex blend of natural odor cues. In insects, the initial processing of odors occurs in the antennal lobe (AL). Afferent peripheral input from olfactory sensory neurons (OSNs) is modified via mostly inhibitory local interneurons (LNs) and transferred by projection neurons (PNs) to higher brain centers. Here we performed optophysiological studies in the AL of the moth, Manduca sexta, and recorded odor-evoked calcium changes in response to antennal stimulation with five monomolecular host volatiles and their artificial mixture. In a double staining approach, we simultaneously measured OSN network input in concert with PN output across the glomerular array. By comparing odor-evoked activity patterns and response intensities between the two processing levels, we show that host mixtures could generally be predicted from the linear summation of their components at the input of the AL, but output neurons established a unique, nonlinear spatial pattern separate from individual component identities. We then assessed whether particularly high levels of signal modulation correspond to behavioral relevance. One of our mixture components, phenyl acetaldehyde, evoked significant levels of nonlinear input-output modulation in observed spatiotemporal activation patterns that were unique from the other individual odorants tested. This compound also accelerated behavioral activity in subsequent wind tunnel tests, whereas another compound that did not exhibit high levels of modulation also did not affect behavior. These results suggest that the high degree of input-output modulation exhibited by the AL for specific odors can correlate to behavioral output.

Inheritance of central neuroanatomy and physiology related to pheromone preference in the male European corn borer.

BACKGROUND: The European corn borer (ECB), Ostrinia nubilalis, is a textbook example of pheromone polymorphism. Males of the two strains (Z and E) prefer opposite ratios of the two pheromone components, Z11- and E11-tetradecenyl acetate, with a sex-linked factor underlying this difference in preference. The male antennal lobes of the two strains contain a pheromone sensitive macroglomerular complex (MGC) that is identical in morphology, but reversed in functional topology. However, hybrids prefer intermediate ratios. How a topological arrangement of two glomeruli can accommodate for an intermediate preference was unclear. Therefore we studied the neurophysiology of hybrids and paternal backcrosses to see which factors correlated with male behavior. RESULTS: Projection neuron (PN) recordings and stainings in hybrids and backcrosses show a dominance of the E-type MGC topology, notwithstanding their intermediate preference. Apparently, the topological arrangement of glomeruli does not directly dictate preference. However, two other factors did correlated very well with preference. First, volumetric measurements of MGC glomeruli demonstrate that, whereas in the parental strains the medial MGC glomerulus is more than 2 times larger than the lateral, in hybrids they are intermediate between the parents, i.e. equally sized. Paternal backcrosses showed that the volume ratio is sex-linked and co-dominant. Second, we measured the summed potential difference of the antennae in response to pheromone stimulation using electroantennogram recordings (EAG). Z-strain antennae responded 2.5 times stronger to Z11 than to E11-14:OAc, whereas in E-strain antennae the ratio was approximately equal. Hybrid responses were intermediate to the parents, and also here the antennal response of the paternal backcrosses followed a pattern similar to the behavioral phenotype. We found no differences in frequency and types of projection and local interneurons encountered between the two strains and their hybrids. CONCLUSIONS: Male pheromone preference in the ECB strains serves as a strong prezygotic reproductive isolation mechanism, and has contributed to population divergence in the field. Our results demonstrate that male pheromone preference is not directly affected by the topological arrangement of olfactory glomeruli itself, but that male preference may instead be mediated by an antennal factor, which causes the MGC glomeruli to be differentially sized. We postulate that this factor affects readout of blend information from the MGC. The results are an illustration of how pheromone preference may be 'spelled out' in the ALs, and how evolution may modulate this.

Identification of female-produced sex pheromone of the honey locust gall midge, Dasineura gleditchiae.

The honey locust gall midge, Dasineura gleditchiae Osten Sacken 1866 (Diptera: Cecidomyiidae) is the main pest of ornamental varieties of the honey locust tree, Gleditsia triacanthos L., in North America, and is now becoming a pest of concern in Europe. Female midges were observed to emerge in the early morning with their ovipositor extended until they mated. Volatiles were collected from virgin females in a closed-loop stripping apparatus and analyzed by gas chromatography (GC) coupled to electroantennographic (EAG) recording from the antenna of a male midge. A single EAG response was observed, which was assumed to be to the major component of the female sex pheromone. This was identified as (Z)-2-acetoxy-8-heptadecene by comparison of its mass spectrum and GC retention times on different columns with those of synthetic standards and by micro-analytical reactions. This compound was synthesized, and the individual enantiomers were produced by kinetic resolution with lipase from Candida antarctica. Analysis of the naturally-produced compound on a cyclodextrin GC column indicated it was the (R)-enantiomer. In EAG dose-response measurements, the (R)-enantiomer alone or in the racemic mixture evoked significant responses from the antennae of male D. gleditchiae, whereas the (S)-enantiomer did not. In field trapping tests, the (R)-enantiomer attracted male D. gleditchiae. The racemic compound was equally attractive, but the (S)-enantiomer was not attractive. Both the pure (R)-enantiomer or racemic (Z)-2-acetoxy-8-heptadecene, applied to red rubber septa in a dose range of 3-30 microg, constitute a strongly attractive bait in sticky traps for monitoring the flight of D. gleditchiae.

Reversed functional topology in the antennal lobe of the male European corn borer.

The European corn borer Ostrinia nubilalis (Hübner) is a model of evolution of sexual communication in insects. Two pheromone strains produce and respond to opposite ratios of the two pheromone components, Z11 and E11-tetradecenylacetate. The Z-strain uses a ratio of 97:3 of Z11:E11 tetradecenylacetate, whereas the E-strain uses a ratio of 1:99. We studied how the difference in male preference correlates with differences in wiring of olfactory input and output neurons in the antennal lobe (AL). Activity-dependent anterograde staining, intracellular recording and immunocytochemistry were used to establish the structure and function of male olfactory receptor neurons (ORNs) and AL projection neurons (PNs). Physiologically characterized neurons were reconstructed using confocal microscopy of alpha-synapsin stained ALs. The ALs of males and females in both strains had approximately 64 glomeruli. In males the macroglomerular complex (MGC) was morphologically similar in the two strains and consisted of two major compartments, a large, medial compartment folded around a smaller, lateral one. Extensive physiological and morphological analysis revealed that in both strains the major pheromone component-specific ORNs and PNs arborize in the medial MGC glomerulus, whereas those sensitive to the minor pheromone component arborize in the lateral glomerulus. In other words, the two strains have an indistinguishable MGC morphology, but a reversed topology. Apparently, the single-gene-mediated shift that causes a radical change in behavior is located upstream of the antennal lobes, i.e. at the ORN level.