Zsolt Kárpáti

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

SUMMARY 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 α-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.

Shifts in sensory neuron identity parallel differences in pheromone preference in the European corn borer

Pheromone communication relies on highly specific signals sent and received between members of the same species. However, how pheromone specificity is determined in moth olfactory circuits remains unknown. Here we provide the first glimpse into the mechanism that generates this specificity in Ostrinia nubilalis. In Ostrinia nubilalis it was found that a single locus causes strain-specific, diametrically opposed preferences for a 2-component pheromone blend. Previously we found pheromone preference to be correlated with the strain and hybrid-specific relative antennal response to both pheromone components. This led to the current study, in which we detail the underlying mechanism of this differential response, through chemotopically mapping of the pheromone detection circuit in the antenna. We determined that both strains and their hybrids have swapped the neuronal identity of the pheromone-sensitive neurons co-housed within a single sensillum. Furthermore, neurons that mediate behavioral antagonism surprisingly co-express up to five pheromone receptors, mirroring the concordantly broad tuning to heterospecific pheromones. This appears as possible evolutionary adaptation that could prevent cross attraction to a range of heterospecific signals, while keeping the pheromone detection system to its simplest tripartite setup.

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.

Flight Tunnel Responses of Z Strain European Corn Borer Females to Corn and Hemp Plants

Abstract European corn borer females Ostrinia nubilalis Hübner (Lepidoptera, Pyralidae) exhibited upwind flight to three main host plants, corn Zea mays, hemp Cannabis sativa and hop Humulus lupulus, in a laboratory wind tunnel. Within a 15-min experimental period, 22.9% to 24.3% mated females flew toward and landed on a single potted corn plant. A potted hemp plant attracted 25.7% females. In a choice test, significantly more females landed on a hemp plant than on an adjacent corn plant. In contrast, paprika Capsicum annuum did not elicit attraction. Headspace collections from corn, hemp, and hop contained 18 compounds which consistently elicited a response from female antennae. Four of these, (Z)-3-hexenyl acetate, β-caryophyllene, (E)-β-farnesene, and (E,E)-α-farnesene co-occurred in three host plants studied. A 4-component blend of these compounds did not attract female moths in the wind tunnel. Availability of a wind tunnel bioassay is, however, a step toward the identification of plant volatiles guiding long-range attraction of gravid corn borer females.

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.

Identification of polyenic hydrocarbons from the northern winter moth, Operophtera fagata , and development of a species specific lure for pheromone traps

Summary.In order to elucidate the composition of the female sex pheromone of the northern (beech) winter moth, Operophtera fagata Scharf. (Lepidoptera: Geometridae), ovipositor extracts of unmated, calling females were analysed by gas chromatography with simultaneous electroantennographic and flame ionization detection (GC-EAD/FID). Male antennal responses indicated three active components, two of which had distinct matching peaks in the FID trace. Using coupled gas chromatography- mass spectrometry (GC-MS), these two compounds were identified as (9Z)-nonadecene (9Z-19:Hy), and (6Z,9Z)-nonadecadiene (6Z9Z-19:Hy), respectively. The third component, present in very small amounts only, was identified as (1,3Z,6Z,9Z)-nonadecatetraene (1,3Z6Z9Z-19:Hy), known as the sex pheromone of the common winter moth, O. brumata. Field tests revealed that traps baited with 6Z9Z-19:Hy and 1,3Z6Z9Z-19:Hy caugth large numbers of male O. fagata. Both compounds were found to be essential for attraction of O. fagata. In addition, the diene prevented captures of co-occurring O. brumata. In contrast, 9Z-19:Hy neither influenced the attractiveness of the two-component mixture towards O. fagata nor contributed to bait specificity. A binary mixture of 6Z9Z-19:Hy and 1,3Z6Z9Z-19:Hy in a ratio of 10:1, applied to pieces of rubber tubing, constituted a highly attractive and species-specific bait for O. fagata, which can be used for monitoring of the flight of this defoliator pest of deciduous forests.

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 μg, constitute a strongly attractive bait in sticky traps for monitoring the flight of D. gleditchiae.

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.

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.

Synthetic blend of larval frass volatiles repel oviposition in the invasive box tree moth, Cydalima perspectalis

Insects find their oviposition sites using visual, contact and olfactory cues. Volatile stimuli emitted by an intact or herbivore-occupied host plant, non-host plants or the herbivore itself can all influence the final decision of females concerning where to lay eggs. Volatile substances surrounding larval excreted pellets, i.e., frass of the invasive box tree moth (Cydalima perspectalis Walker) were collected, and the physiological activity was investigated by coupled gas chromatographic–electroantennographic detection. Based on structural elucidation, two aromatic derivates and one terpene alcohol were identified to be physiologically active on the antennae of the adults: guaiacol, (±)-linalool and veratrol. For all compounds, antennal responses were found to be dose dependent with EAG amplitudes being the highest at the highest dose levels. Females were also more sensitive to all three compounds compared to males. Single sensillum recordings on mated female antennae revealed that these frass compounds triggered 22% of the tested olfactory sensory neurons housed in trichoid sensilla. Behavioral bioassays indicated that the blend of these compounds had an oviposition-repellent effect on conspecific females: individuals laid significantly fewer eggs on boxwood plants equipped with dispensers loaded with the synthetic blend compared to those treated with natural frass or the control plants. This difference likely originated from the measured rapid changes in the volatile profile of larval excrement when exposed to the air at room temperature. Our findings have the potential to unravel the complex ecology of this invasive moth species characterized by rapid range expansion and extensive damage in Europe.