In-vivo two-photon imaging of the honey bee antennal lobe

Due to the honey bee's importance as a simple neural model, there is a great need for new functional imaging modalities. Herein we report on the use of two-photon microscopy for in-vivo functional and morphological imaging of the honey bee's olfactory system focusing on its primary centers, the antennal lobes (ALs). Our imaging platform allows for simultaneously obtaining both morphological measurements of the AL and in-vivo calcium recording of neural activities. By applying external odor stimuli to the bee's antennas, we were able to record the characteristic odor response maps. Compared to previous works where conventional fluorescence microscopy is used, our approach offers all the typical advantages of multi-photon imaging, providing substantial enhancement in both spatial and temporal resolutions while minimizing photo-damages and autofluorescence contribution with a four-fold improvement in the functional signal. Moreover, the multi-photon associated extended penetration depth allows for functional imaging within profound glomeruli.Comment: 3 pages, 3 figure

Feeding regulates sex pheromone attraction and courtship in Drosophila females

In Drosophila melanogaster, gender-specific behavioural responses to the male-produced sex pheromone cis-vaccenyl acetate (cVA) rely on sexually dimorphic, third-order neural circuits. We show that nutritional state in female flies modulates cVA perception in first-order olfactory neurons. Starvation increases, and feeding reduces attraction to food odour, in both sexes. Adding cVA to food odour, however, maintains attraction in fed females, while it has no effect in males. Upregulation of sensitivity and behavioural responsiveness to cVA in fed females is paralleled by a strong increase in receptivity to male courtship. Functional imaging of the antennal lobe (AL), the olfactory centre in the insect brain, shows that olfactory input to DA1 and VM2 glomeruli is also modulated by starvation. Knocking down insulin receptors in neurons converging onto the DA1 glomerulus suggests that insulin-signalling partly controls pheromone perception in the AL, and adjusts cVA attraction according to nutritional state and sexual receptivity in Drosophila females

Lateralization in the Invertebrate Brain: Left-Right Asymmetry of Olfaction in Bumble Bee, Bombus terrestris

Brain and behavioural lateralization at the population level has been recently hypothesized to have evolved under social selective pressures as a strategy to optimize coordination among asymmetrical individuals. Evidence for this hypothesis have been collected in Hymenoptera: eusocial honey bees showed olfactory lateralization at the population level, whereas solitary mason bees only showed individual-level olfactory lateralization. Here we investigated lateralization of odour detection and learning in the bumble bee, Bombus terrestris L., an annual eusocial species of Hymenoptera. By training bumble bees on the proboscis extension reflex paradigm with only one antenna in use, we provided the very first evidence of asymmetrical performance favouring the right antenna in responding to learned odours in this species. Electroantennographic responses did not reveal significant antennal asymmetries in odour detection, whereas morphological counting of olfactory sensilla showed a predominance in the number of olfactory sensilla trichodea type A in the right antenna. The occurrence of a population level asymmetry in olfactory learning of bumble bee provides new information on the relationship between social behaviour and the evolution of population-level asymmetries in animals

Putative Chemosensory Receptors of the Codling Moth, Cydia pomonella, Identified by Antennal Transcriptome Analysis

The codling moth, Cydia pomonella, is an important fruit pest worldwide. As nocturnal animals, adults depend to a large extent on olfactory cues for detection of food and mates, and, for females, oviposition sites. In insects, odor detection is mediated by odorant receptors (ORs) and ionotropic receptors (IRs), which ensure the specificity of the olfactory sensory neuron responses. In this study, our aim was to identify chemosensory receptors in the codling moth as a means to uncover new targets for behavioral interference. Using next-generation sequencing techniques, we identified a total of 43 candidate ORs, one gustatory receptor and 15 IRs in the antennal transcriptome. Through Blast and sequence similarity analyses we annotated the insect obligatory co-receptor ORco, five genes clustering in a conserved clade containing sex pheromone receptors, one homolog of the Bombyx mori female-enriched receptor BmorOR30 (but no homologs of the other B. mori female-enriched receptors) and one gene clustering in the sugar receptor family. Among the candidate IRs, we identified homologs of the two highly conserved co-receptors IR8a and IR25a, and one homolog of an IR involved in phenylethyl amine detection in Drosophila. Our results open for functional characterization of the chemosensory receptors of C. pomonella, with potential for new or refined applications of semiochemicals for control of this pest insect

A multimodal approach for tracing lateralization along the olfactory pathway in the honeybee through electrophysiological recordings, morpho-functional imaging, and behavioural studies

Recent studies have revealed asymmetries between the left and right sides of the brain in invertebrate species. Here we present a review of a series of recent studies from our labs, aimed at tracing asymmetries at different stages along the honeybee's (Apis mellifera) olfactory pathway. These include estimates of the number of sensilla present on the two antennae, obtained by scanning electron microscopy, as well as electroantennography recordings of the left and right antennal responses to odorants. We describe investigative studies of the antennal lobes, where multi-photon microscopy is used to search for possible morphological asymmetries between the two brain sides. Moreover, we report on recently published results obtained by two-photon calcium imaging for functional mapping of the antennal lobe aimed at comparing patterns of activity evoked by different odours. Finally, possible links to the results of behavioural tests, measuring asymmetries in single-sided olfactory memory recall, are discussed.Comment: 28 pages, 8 figure

Comparative behavioural and anatomo-pathological investigations on Musca domestica L. adults treated with a new strain of Bacillus sp. close related to B. Thuringiensis (Berliner)

Comparative behavioural bioassays and anatomo-pathological investigations have been carried out on Musca domestica adults to test insecticidal activity of a new isolate of Bacillus sp. (characterization in course) close related to B. thuringiensis (Berliner). Both behavioural bioassays and anatomo-pathological tests, parallel the results previously reported for many other insects similarly treated with different strains of the well known B. thuringiensis. Flies fed on a suspension of a Bacillus sp. new isolate sporulated culture, display progressive sluggish and shaky behaviour, decreased responsiveness to external stimuli, gradual feeding inhibition, and general paralysis, until they die (over 52% within 72 hrs, and almost all specimens within 4-5 days, after treatment). Anatomo-histological and ultrastructural investigations corroborate the results from behavioural bioassays, proving that flies treated with Bacillus sp. undergo progressive degeneration (up to rupture) of midgut and malpighian tubules’ epithelial cells, as well as of the midgut muscular sheath, as reported for other insect species treated with B. thuringiensis. Comparative tests on M. domestica adults treated with analogous suspension of B. th. var. kurstaki (HD-1) gave rather negative results. Key words: behavioural bioassays, electron microscopy, gut anatomy, insecticidal activity, midgut histology, light microscopy. RICERCHE COMPORTAMENTALI ED ANATOMO-PATOLOGICHE SU ADULTI DI MUSCA DOMESTICA L. TRATTATI CON UN NUOVO ISOLATO DI BACILLUS SP. AFFINE AL B. THURINGIENSIS (BERLINER). Biosaggi comportamentali e indagini anatomo-patologiche sono state parallelamente condotte su adulti di Musca domestica L. per provare l’attività insetticida di un nuovo isolato di Bacillus sp. (attualmente in via di caratterizzazione), affine al rinomato Bacillus thuringiensis (Berliner). Sia le prove comportamentali che quelle anatomo-istologiche ed ultrastrutturistiche hanno dato risultati simili a quelli riportati in letteratura per vari altri insetti trattati analogamente con diverse varietà di B. thuringiensis. Mosche alimentate con una sospensione della coltura sporulata di Bacillus sp. vanno incontro progressivamente a intorpidimento generale e tremore, ridotta sensibilità a stimoli esterni, graduale rifiuto del cibo e paralisi generale, fino alla morte (oltre 52% delle mosche muoiono entro 72 ore, e quasi tutte in 4-5 giorni dalla prima somministrazione della sospensione batterica). Le osservazioni anatomo-istologiche ed ultrastrutturistiche corroborano i risultati dei biosaggi comportamentali, evidenziando nelle mosche trattate col nuovo isolato di Bacillus sp. una progressiva degenerazione delle cellule epiteliali del mesentero e dei tubi malpighiani, come pure della tunica muscolare del mesentero, come è stato visto per altri insetti trattati con B. thuringiensis (Berliner). Prove di confronto con adulti di M. domestica analogamente trattati con una sospensione di B. th var. kurstaki (HD-1) hanno dato risultati sostanzialmente negativi. Parole chiave: anatomia intestinale, attività insetticida, biosaggi comportamentali, istologi

Morpho-functional asymmetry of the olfactory receptors of the honeybee (Apis mellifera)

Lateralization, i.e., the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate species and, recently, in invertebrate species as well. In the Honeybee, Apis mellifera L. (Hymenoptera Apidae), it has been shown that short-term (<1 h) recall of olfactory memories would be possible mainly from the right rather than from the left antenna. Here we confirmed this finding showing that recall of the olfactory memory 1 h after training to associate (−)-linalool, a floral volatile compound, with a sugar reward, as revealed by the bee extending its proboscis when presented with the trained odour, was better when the odour was presented to the right rather than to the left antenna. We then measured the number of sensilla present on the left and right antenna by scanning electron microscopy. Results showed that putative olfactory sensilla (placodea, trichodea, basiconica) were significantly more abundant on the right antenna surface than on the left antenna surface, whereas sensilla not involved in olfaction (campaniformia, coeloconica and chaetica) tended to be more abundant on the left than on the right antenna surface

Neural coding merges sex and habitat chemosensory signals in an insect herbivore.

Understanding the processing of odour mixtures is a focus in olfaction research. Through a neuroethological approach, we demonstrate that different odour types, sex and habitat cues, are coded together in an insect herbivore. Stronger flight attraction of codling moth males, Cydia pomonella, to blends of female sex pheromone and plant odour, compared with single compounds, was corroborated by functional imaging of the olfactory centres in the insect brain, the antennal lobes (AL). The macroglomerular complex (MGC) in the AL, which is dedicated to pheromone perception, showed an enhanced response to blends of pheromone and plant signals, while the response in glomeruli surrounding the MGC was suppressed. Intracellular recordings from AL projection neurons that transmit odour information to higher brain centres, confirmed this synergistic interaction in the MGC. These findings underscore that, in nature, sex pheromone and plant odours are perceived as an ensemble. That mating and habitat cues are coded as blends in the MGC of the AL highlights the dual role of plant signals in habitat selection and in premating sexual communication. It suggests that the MGC is a common target for sexual and natural selection in moths, facilitating ecological speciation

The female sex pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster

Specific mate communication and recognition underlies reproduction and hence speciation. Our study provides new insights in Drosophila melanogaster premating olfactory communication. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues. Female-produced, species specific volatile pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone (Z)-11-octadecenyl acetate (cVA) interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males. Two isoforms of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. However, only Z4-11Al mediates courtship in experienced males, not (R)-linalool. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds. Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in contrast to Z4-11Al. A blend of the food odour vinegar and cVA attracted females, while a blend of vinegar and female pheromone Z4-11Al attracted males, instead. Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects

Mean number ± SE of sensilla for the right antenna (white bars) and for the left antenna (grey bars) of <i>Bombus terrestris</i> foragers in function of the segment number.

<p>Putative olfactory sensilla: placodea, trichodea type A, basiconica, coeloconica (upper graphs). Non-olfactory sensilla: trichodea type B, ampullacea (lower graphs). Data were analyzed by ANOVA with antenna, segment and sensilla as within-subjects factor. An overall antenna effect emerged (F_1,13 = 22.56, p<0.001). A significant effect of segment (F_7,91 = 43.20, p<0.001), sensillum type (F_5,65 = 396.40, p<0.001) and antenna per sensillum type interaction (F_5,65 = 17.89, p<0.001) was revealed. Asterisks indicate a significant right antenna dominance in the number of olfactory sensilla trichodea type A (F_1,13 = 21.26, p<0.001). No significant antenna effects were found in the number of sensilla basiconica (F_1,13 = 1.47, p = 0.247), sensilla coeloconica (F_1,13 = 3.61, p = 0.08) and sensilla placodea (F_1,13 = 0.97, p = 0.342). Analyses of non-olfactory sensilla did not reveal any significant difference between right and left antennae in the number of sensilla trichodea type B (F_1,13 = 3.45, p = 0.086) and sensilla ampullacea (F_1,13 = 0.10, p = 0.755).</p