Caste and sex specific olfactory glomerular organization and brain architecture in two sympatric ant species Camponotus sericeus and Camponotus compressus (Fabricius, 1798)

We use monoclonal antibodies against synaptic proteins and anterograde tracing with neurobiotin to describe the architecture of the antennal lobes in different castes of two ant species – Camponotus sericeus and Camponotus compressus. The reproductives and worker classes are readily categorized based on size and external morphology. The overall organization of brain neuropile is comparable between castes with differences only in the visual ganglia. Males have a larger fraction of neuropile occupied by the medulla and lobula than females. In the diurnal species, C. sericeus these regions are more highly represented, than in the nocturnal species C. compressus. The most striking differences are in the antennal lobe where males possess a macroglomerulus, which is about ten times larger in volume than the other glomeruli; such a specialization is absent in females. Minor workers possess a significantly larger number of glomeruli than the majors despite the smaller overall volume of the lobe. These caste-specific differences occur mainly within glomerular clusters that receive input from sensory neurons that project in tracts – T4 and T5 – within the antennal nerve. The comparative anatomy of different castes of ants provides an entry point into a future systematic analysis of how divergent brain architectures can arise within a single species.     

Anatomical organization of antennal-lobe glomeruli in males and females of the scarab beetle Holotrichia diomphalia (Coleoptera: Melolonthidae)

The glomerular organization of the primary olfactory brain center, the antennal lobe, was studied in males and females of Holotrichia diomphalia adults using serial histological sections labeled by the reduced silver-stain technique. The results revealed an apparent sexual dimorphism. Whereas an enlarged cap-shaped glomerulus was found at the antennal nerve entrance into the antennal lobe in males, no such unit was present in females. Also the size of the antennal lobe differed between the sexes, the antennal lobe of males being larger than that of females. We estimated the total number of glomeruli at approximately 60 units in the female antennal lobe. In males, we could discriminate only those glomeruli that were located in the anterior area of the antennal lobe.     

Phenotypic plasticity in number of glomeruli and sensory innervation of the antennal lobe in leaf‐cutting ant workers (A. vollenweideri)

In the leaf‐cutting ant Atta vollenweideri, the worker caste exhibits a pronounced size‐polymorphism, and division of labor is dependent on worker size (alloethism). Behavior is largely guided by olfaction, and the olfactory system is highly developed. In a recent study, two different phenotypes of the antennal lobe of Atta vollenweideri workers were found: MG‐ and RG‐phenotype (with/without a macroglomerulus). Here we ask whether the glomerular numbers are related to worker size. We found that the antennal lobes of small workers contain ～390 glomeruli (low‐number; LN‐phenotype), and in large workers we found a substantially higher number of ～440 glomeruli (high‐number; HN‐phenotype). All LN‐phenotype workers and some small HN‐phenotype workers do not possess an MG (LN‐RG‐phenotype and HN‐RG‐phenotype), and the remaining majority of HN‐phenotype workers do possess an MG (HN‐MG‐phenotype). Using mass‐staining of antennal olfactory receptor neurons we found that the sensory tracts divide the antennal lobe into six clusters of glomeruli (T1–T6). In LN‐phenotype workers, ～50 glomeruli are missing in the T4‐cluster. Selective staining of single sensilla and their associated receptor neurons revealed that T4‐glomeruli are innervated by receptor neurons from the main type of olfactory sensilla, the Sensilla trichodea curvata. The other type of olfactory sensilla (Sensilla basiconica) exclusively innervates T6‐glomeruli. Quantitative analyses of differently sized workers revealed that the volume of T6 glomeruli scales with the power of 2.54 to the number of Sensilla basiconica. The results suggest that developmental plasticity leading to antennal‐lobe phenotypes promotes differences in olfactory‐guided behavior and may underlie task specialization within ant colonies. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 222–234, 2010.     

In vitro culture of hymenopterous larval endoparasitoids

The solitary endoparasitoids Cotesia (= Apanteles) marginiventris and Microplitis croceipes, which attack noctuid larvae, were successfully cultured from post-germband stage eggs to fully grown first-instar larvae in completely artificial (but undefined) media. However, growth rates were slower than in vivo, and while some larvae of each species attained apparent competence to moult (based upon characteristic size and form), none did so.Up to 75% of pregermband M. croceipes eggs developed and hatched when co-cultured with fat body from non-parasitized larvae of the habitual host, Heliothis zea. Heat-treated haemolymph plasma from a non-permissive host, Manduca sexta, also fostered development by ca. 40% of newly laid M. croceipes eggs, but plasma from H. zea larvae was not beneficial. Growth rates of C. marginiventris and M. croceipes larvae were not improved by haemolymph plasma from their habitual hosts (Spodoptera frugiperda and H. zea, respectively).Heat-treated foetal bovine serum was growth-promoting for post-germband eggs and larvae of both parasitoids. Treatment of foetal bovine serum by charcoal adsorption (to remove sterols and peptide hormones) diminished its value as a supplement. Two major proteins of the serum, foetuin and albumin, also promoted survival and growth for both parasitoid species. The potential relevance of these findings to in vitro rearing of endoparasitoids is discussed.     

Morphological and developmental analysis of peripheral antennal chemosensory sensilla and central olfactory glomeruli in worker castes of Camponotus compressus (Fabricius, 1787)

The antennal lobes of different castes of the ant species Camponotus compressus show a marked diversity in the organization of their olfactory glomeruli. Notably, there is a significant difference in the number and size of glomeruli between the reproductives and the workers and among the different worker castes. In this report, we investigate the notion that these caste-specific differences in glomerular number might be accounted for, at least in part, by the differences in numbers of olfactory sensilla that target the antennal lobe. For this, we examine the number of sensilla on the antennal flagella of all the individual castes of C. compressus. This analysis reveals a striking correlation between sensillar number and the number of antennal glomeruli in a given caste. As a first step in investigating the causal mechanisms that might give raise to this correlation, we carry out an initial characterization of olfactory system development in the minor workers of C. compressus. We analyze the temporal pattern of innervations of the developing antennal lobe by olfactory sensory neuron axons. We document the development of the olfactory glomeruli in the antennal lobe during this process, which occurs during early pupal stages. Our findings provide the basis for future manipulative developmental studies on the role of sensory afferent number in glomerular development of different castes within the same species.     

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.     

Transplant Antennae and Host Brain Interact to Shape Odor Perceptual Space in Male Moths

Behavioral responses to odors rely first upon their accurate detection by peripheral sensory organs followed by subsequent processing within the brain’s olfactory system and higher centers. These processes allow the animal to form a unified impression of the odor environment and recognize combinations of odorants as single entities. To investigate how interactions between peripheral and central olfactory pathways shape odor perception, we transplanted antennal imaginal discs between larval males of two species of moth Heliothis virescens and Heliothis subflexa that utilize distinct pheromone blends. During metamorphic development olfactory receptor neurons originating from transplanted discs formed connections with host brain neurons within olfactory glomeruli of the adult antennal lobe. The normal antennal receptor repertoire exhibited by males of each species reflects the differences in the pheromone blends that these species employ. Behavioral assays of adult transplant males revealed high response levels to two odor blends that were dissimilar from those that attract normal males of either species. Neurophysiological analyses of peripheral receptor neurons and central olfactory neurons revealed that these behavioral responses were a result of: 1. the specificity of H. virescens donor olfactory receptor neurons for odorants unique to the donor pheromone blend and, 2. central odor recognition by the H. subflexa host brain, which typically requires peripheral receptor input across 3 distinct odor channels in order to elicit behavioral responses.     

Anatomical and neurochemical classification of the antennal glomeruli in Drosophila melanogaster meigen (Diptera : Drosophilidae)

We are interested in how information about odours is processed in the antennal lobe of Drosophila melanogaster Meigen (Diptera : Drosophilidae). Our ultimate aim is to study how genes specify the development and functioning of the olfactory pathway. Such studies require a thorough knowledge of the anatomy, neurochemistry and development of the antennal system in normal files. This paper is a first attempt towards this end. Toluidine-methylene blue staining has revealed the presence of 22 glomeruli in the antennal lobe of Drosophila melanogaster. The number and size of glomeruli are invariant between individuals and the sexes. Fifteen of the glomeruli demonstrated specific uptake of choline, which is suggestive of some cholinergic innervation. In developmental studies, all glomeruli were found to be morphologically mature by 72 hr after formation of the white pupa. This study is a necessary first step towards an investigation of the role played by the glomeruli as functional units for odour coding.     

Comparison of detection ability of learned odors between males and females in the larval parasitoid Microplitis croceipes

Although female parasitic wasps are known to learn to associate odors with hosts and food, the ability of males to learn and detect odors has been neglected. We conducted laboratory experiments to compare the detection ability of learned odors between males and females in the larval parasitoid Microplitis croceipes (Cresson) (Hymenoptera: Braconidae). We first conditioned males and females to associate sucrose water with methyl benzoate, 3‐octanone, or cyclohexanone, and then observed their behavior toward various concentrations (40 ng l^?1?4 mg l^?1) of the trained odors. Conditioned male wasps responded as well as female wasps to various concentrations of the three odors. Response times by wasps to these three odors were not significantly different between sexes. For the three odors, response times of both sexes were longer at the intermediate concentrations (40–400 µg l^?1) than the higher or lower concentrations. The present study suggests that M. croceipes males can learn and respond to the three chemicals tested as well as the females, and conditioned males are as sensitive to learned odors as conditioned females. By using their sensitive learning and odor‐detection capabilities, M. croceipes males could search for food sources as efficiently as females under natural conditions.     

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.     

Caste- and sex-specific adaptations within the olfactory pathway in the brain of the ant Camponotus floridanus

Olfaction plays a key role in mediating ant behavior, and ant societies are characterized by caste- and sex-specific division of labor. We propose that caste- and sex-specific adaptations in the olfactory pathway promote differences in olfactory behavior. This study compares olfactory centers in the brain of large (major) workers, small (minor) workers, virgin queens, and males of the carpenter ant Camponotus floridanus. The number of glomeruli in the antennal lobe was similar in the female castes, although the glomerular volumes differed. Males had approximately 45% fewer glomeruli compared to females (approximately 258 and approximately 434) and one antennal sensory tract was absent. A dual output pathway to the mushroom bodies was present in males. In contrast to females, however, the number of glomeruli connected to the medial antennocerebral tract was substantially smaller than those associated with the lateral tract. All glomeruli in the male antennal lobe contained serotonergic processes, whereas in the female castes glomeruli in the large tract six cluster lacked serotonergic innervations. We conclude that differences in general glomerular organization are subtle among the female castes, but sex-specific differences in the number, connectivity and neuromodulatory innervation of glomeruli are substantial and likely to underlie differences in olfactory processing and learning.     

Olfactory subsystems in the honeybee: sensory supply and sex specificity

The antennae of honeybee (Apis mellifera) workers and drones differ in various aspects. One striking difference is the presence of Sensilla basiconica in (female) workers and their absence in (male) drones. We investigate the axonal projection patterns of olfactory receptor neurons (ORNs) housed in S. basiconica in honeybee workers by using selective anterograde labeling with fluorescent tracers and confocal-microscopy analysis of axonal projections in antennal lobe glomeruli. Axons of S. basiconica-associated ORNs preferentially projected into a specific glomerular cluster in the antennal lobe, namely the sensory input-tract three (T3) cluster. T3-associated glomeruli had previously been shown to be innervated by uniglomerular projection (output) neurons of the medial antennal lobe tract (mALT). As the number of T3 glomeruli is reduced in drones, we wished to determine whether this was associated with the reduction of glomeruli innervated by medial-tract projection neurons. We retrogradely traced mALT projection neurons in drones and counted the innervated glomeruli. The number of mALT-associated glomeruli was strongly reduced in drones compared with workers. The preferential projections of S. basiconica-associated ORNs in T3 glomeruli together with the reduction of mALT-associated glomeruli support the presence of a female (worker)-specific olfactory subsystem that is partly innervated by ORNs from S. basiconica and is associated with the T3 cluster of glomeruli and mALT projection neurons. We propose that this olfactory subsystem supports parallel olfactory processing related to worker-specific olfactory tasks such as the coding of colony odors, colony pheromones and/or odorants associated with foraging on floral resources.     

Neuronal architecture of the antennal lobe in Drosophila melanogaster

Summary Computer reconstruction of the antennal lobe of Drosophila melanogaster has revealed a total of 35 glomeruli, of which 30 are located in the periphery of the lobe and 5 in its center. Several prominent glomeruli are recognizable by their location, size, and shape; others are identifiable only by their positions relative to prominent glomeruli. No obvious sexual dimorphism of the glomerular architecture was observed. Golgi impregnations revealed: (1) Five of the glomeruli are exclusive targets for ipsilateral antennal input, whereas all others receive afferents from both antennae. Unilateral amputation of the third antennal segment led to a loss of about 1000 fibers in the antennal commissure. Hence, about 5/6 of the approximately 1200 antennal afferents per side have a process that extends into the contralateral lobe. (2) Afferents from maxillary palps (most likely from basiconic sensilla) project into both ipsi-and contralateral antennal lobes, yet their target glomeruli are apparently not the same as those of antennal basiconic sensilla. (3) Afferents in the antennal lobe may also stem from pharyngeal sensilla. (4) The most prominent types of interneurons with arborizations in the antennal lobe are: (i) local interneurons ramifying in the entire lobe, (ii) unilateral relay interneurons that extend from single glomeruli into the calyx and the lateral protocerebrum (LPR), (iii) unilateral interneurons that connect several glomeruli with the LPR only, (iv) bilateral interneurons that link a small number of glomeruli in both antennal lobes with the calyx and LPR, (v) giant bilateral interneurons characterized by extensive ramifications in both antennal lobes and the posterior brain and a cell body situated in the midline of the suboesophageal ganglion, and (vi) a unilateral interneuron with extensive arborization in one antennal lobe and the posterior brain and a process that extends into the thorax. These structural results are discussed in the context of the available functional and behavioral data.Abbreviations AC antennal commissure - AMMC antennal mechanosensory and motor center - iACT, mACT, oACT inner/middle/outer antenno-cerebral tract - bACTI, uACTI bilateral/unilateral ACT relay interneuron - AN antennal nerve - AST antenno-suboesophageal tract - FAI fine arborization relay interneuron - GSI giant symmetric relay interneuron - LI local interneuron - LPR lateral protocerebrum - SOG suboesophageal ganglion - TI thoracic relay interneuron - bVI bilateral V-relay interneuron     

Aldehyde oxidases and dehydrogenases in antennae of five moth species

Conversion of Z11–16:A1 to its corresponding carboxylic acid was examined in male antennal extracts from Heliothis subflexa, Heliothis virescens, Heliothis zea, Manduca sexta and Spodoptera frugiperda moths. Both aldehyde dehydrogenase (ALDH) and aldehyde oxidase (AO) activities were detected by radiochromatographic assays using [³H]Z11–16:A1 as the substrate and by spectroscopic assays using Z11–16:A1 as the substrate. AO activity in each of the five moths showed a broad substrate specificity which included C₄ to C₁₈ aliphatic aldehydes and benzaldehyde.ALDH and AO enzymes from the male and female antennal extracts were identified after electrophoretic protein separation. AO enzymes were visualized on native polyacrylamide gels with formazan- or horseradish peroxidase-mediated stain coupled to the AO-catalyzed oxidation of benzaldehyde. Both sexes of most species showed intense AO-stained bands of similar mobility. Molecular subunits of potential ALDH enzymes were visualized by fluorography of SDS-PAGE-separated proteins after covalent modification by [³H](Z)-1,11-hexadecadien-3-one, a selective affinity label for ALDH. ALDH subunits appeared at 51 ± 1 kDa in antennae from males of all five species and females of three species; female H. zea and M. sexta moths lacked these characteristic ALDH subunits. These results constitute the first biochemical comparisons of coexisting ALDH and AO enzymes in antennal extracts from lepidopteran species from two families.     

Larval chemosensory projections and invasion of adult afferents in the antennal lobe of Drosophila

We have studied the fate of olfactory afferents during metamorphic transformation of Drosophila melanogaster. Intracellular labeling of afferents from larval head chemosensilla suggests that the larval antennal lobe may be an olfactory target, whereas tritocerebral and suboesophageal centers are likely targets of gustatory sensilla. Application of monoclonal antibody 22C10 shows that the larval antennal nerve is the precursor of the adult antennal nerve and is used as a centripetal pathway for the adult afferents. Likely guidance cues are larval olfactory afferents that persist during early metamorphosis. P[GAL4] enhancer trap lines are introduced as efficient markers to follow the establishment of adult sensory projections. β-Galactosidase and the bovine TAU protein were used as reporter proteins, and their expression patterns are compared. P[GAL4] lines MT14 and KL116 demonstrate that adult antennal afferents have arrived in the antennal lobe 24 h after pupariation and extend to the contralateral lobe 6 h later. Line MT14 expresses GAL4 mostly in basiconic sensilla and in certain trichoid sensilla, whereas KL116 is specific for trichoid and a small subset of basiconic sensilla. In the antennal lobe, largely complementary subsets of glomeruli are labeled by the two lines, in agreement with the observation that particular types of sensilla project to particular target glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 281–297, 1997.     

Egg Load and Body Size of Lab-cultured Cotesia marginiventris

The egg load of lab-cultured Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae), a solitary koinobiont endoparasitoid of noctuid caterpillars, was determined in this study. Information on egg load may provide clues to more efficient in vivo rearing of C. marginiventris. I tested the hypothesis that egg load, defined as the number of mature oöcytes (i.e., fully chorionated eggs) found in adult females, was related to body size. Cotesia marginiventris females possessed two ovaries and two ovarioles per ovary; mature eggs were found in ovaries and oviducts. Newly-emerged females held an average of 149 mature eggs. Immature eggs were slightly visible in the distal portions of the ovarioles; they were not counted. Egg load was marginally related to body size (i.e., hind tibia length). The results of this study suggest that (1) body size can sometimes predict egg load or potential fecundity of lab-cultured C. marginiventris and (2) an efficient rearing system that exploits the potential fecundity of C. marginiventris might involve using young females and allowing them to oviposit in new hosts, each day, for up to a week.     

Interspecific discrimination and larval competition amongMicroplitis croceipes,Microplitis demolitor,Cotesia kazak (Hym.: Braconidae), andHyposoter didymator (Hym.: Ichneumonidae), parasitoids ofHeliothis virescens (Lep.: Noctuidae)

Interspecific host discrimination by adults, and larval competition among the endoparasitoidsMicroplitis croceipes (Cresson),Microplitis demolitor Wilkinson,Cotesia kazak (Telenga) andHyposoter didymator (Thunberg) were investigated usingHeliothis virescens (F.) as the host. In ovipositional choice tests, the mean number of encounters and ovipositions for unparasitized hosts was not significantly different from the mean number of encounters and ovipositions for parasitized hosts for each treatment combination (P>0.05). Thus, none of the parasitoid species discriminated between host larvae recently parasitized once by a female of another species und unparasitized hosts. However, in all but two cases, females did discriminate between unparasitized hosts and hosts in which an early first instar of the first-attacking species was developing.Cotesia kazak andH. didymator did not discriminate between unparasitized hosts and hosts parasitized by an early first instar ofM. demolitor. Larval competition among these parasitoid species was studied for three time intervals between the first and second species parasitization: 1) second species attack immediately (5–15 sec) after the first; 2) second species attack 24 h after the first; and 3) second species attack 48 h after the first. Time until egg eclosion was shortest forM. demolitor, thenC. kazak, thenM. croceipes, and longest forH. didymator. When the second parasitoid species attacked a host immediately after the first species, the species in which egg eclosion occurred first was the victor more frequently, except whenM. demolitor competed withC. kazak andH. didymator. With a 24 h delay between the first and second species to attack, the older first instar from the first parasitization usually outcompeted the younger first instar from the second attack. A first instar from the second species to attack generally outcompeted the second instar of the first species when the second parasitization had been delayed 48 h. Competiors were eliminated mainly by physical attack, butC. kazak andM. croceipes apparently also killedH. didymator eggs by physiological processes.     

Spatial organization of the antennal lobe in Cylindroiulus punctatus (Leach) (Myriapoda : Diplopoda)

The topography of the antennal afferent pathway has been studied in detail for the first time in a myriapod, Cylindroiulus punctatus, by cellular staining, following localized application of cobalt chloride to the cut end of one antenna. At the lateral entrance of the sensory antennal nerve into the deutocerebrum, 3 main groups of tracts have been distinguished. In the antennal lobe, there are two groups of glomeruli: a ventral group of 3 glomeruli and a dorsal group of 15 or 16 glomeruli. The number, position and dimensions of the glomeruli indicate that the glomerular organization is globally invariant (5 individuals).     

Physiology and antennal lobe projections of olfactory receptor neurons from sexually isomorphic sensilla on male <Emphasis Type="Italic">Heliothis virescens</Emphasis>

The neurophysiology and antennal lobe projections of olfactory receptor neurons (ORNs) within sexually isomorphic short trichoid sensilla of male Heliothis virescens (Noctuidae: Lepidoptera) were investigated using cut-sensillum recording and cobalt-lysine staining. A total of 202 sensilla were sorted into 14 possible sensillar categories based on odor responses and physiology of ORNs within. Seventy-two percent of the sensilla identified contained ORNs stimulated by conspecific odors. In addition, a large number of ORNs were specifically sensitive to ß-caryophyllene, a plant-derived volatile (N = 41). Axons originating from ORNs associated with individual sensilla were stained with cobalt lysine (N = 67) and traced to individual glomeruli in the antennal lobe. ORNs with responses to female sex pheromone components exhibited similar axonal projections as those previously described from ORNs in long sensilla trichodea in male H. virescens. Antennal lobe axonal arborizations of ORNs sensitive to hairpencil components were also located in glomeruli near the base of the antennal nerve, whilst those sensitive to plant odorants projected to more medial glomeruli. Comparisons with ORNs described from female H. virescens supports the notion that glomeruli at the base of the antennal nerve are associated with conspecific and interspecific odorants, whereas those located medially are associated with plant volatiles.     

Three-dimensional antennal lobe atlas of the oriental fruit moth, <Emphasis Type="Italic">Cydia molesta</Emphasis> (Busck) (Lepidoptera: Tortricidae): comparison of male and female glomerular organization

The oriental fruit moth Cydia molesta is an important pest and the behavioural role of olfactory signals such as pheromones and plant volatiles have been studied extensively in both sexes. To understand odour processing further, however, detailed knowledge of the anatomy of the olfactory system is crucial. In the present study, an atlas of the antennal lobe (AL) is presented based on the three-dimensional reconstructions of both ALs of three male and three female brains by means of neuroanatomical and computational approaches. We identified 48–49 "ordinary" glomeruli and one large glomerulus situated at the entrance of the antennal nerve in males, and 49–52 "ordinary" glomeruli and one large glomerulus in the ventro-medial part of the AL in females. Anomalous supernumerary, anomalous missing and sexually dimorphic glomeruli were found in the studied individuals in greater numbers than in other lepidopteran species. Male and female maps were compared with respect to glomerular size and position with 45 glomeruli being matched, indicating a conserved glomerular pattern between the sexes. Three additional glomeruli were sexually dimorphic in size and five male-specific and six female-specific glomeruli were also found. Palp backfills resulted in the staining of a unique glomerulus in both sexes identified as the sexually dimorphic glomerulus 45. This glomerulus was never stained from antennal backfills, which stained the other glomeruli of the AL. The three-dimensional atlas can now be used to elucidate the functional role of individual glomeruli in both sexes of C. molesta. Nélia Varela and Louise Couton contributed equally to this work. This study was supported by research grants from INRA (Projet Jeune Equipe and Projet S.P.E.) to S.A. and J.P.R. and by the Spanish Ministry of Science and Technology (research grant AGL2004-05812/AGR to J.A. and C.G., Spanish Science and Education Department, MEC). N.V. was financed by fellowship no. BES-2005-7605 (MEC, Spain).