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.     

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.     

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.     

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.     

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.     

Species and sexual differences in antennal lobe architecture and glomerular organization in two parasitoids with different degree of host specificity, Microplitis croceipes and Cotesia marginiventris

The endoparasitic wasps (Hymenoptera: Braconidae), Microplitis croceipes (specialist) and Cotesia marginiventris (generalist), are parasitoids of lepidopteran larvae and differ in their degree of host specificity. Recent studies have reported key differences between the two species in the abundance of antennal olfactory sensilla and their response to host-related volatiles. Here, we have compared antennal lobe architecture and glomerular organization in the two parasitoid species by using a combination of axonal tract tracing techniques and confocal microscopy. In M. croceipes, the medial half of the antennal lobe is larger with a greater number of glomeruli compared with the lateral half, whereas in C. marginiventris, the lateral half is larger than the median half. The volume of the antennal lobe is approximately 2.5 times greater in M. croceipes than in C. marginiventris. However, the number of glomeruli per antennal lobe is only slightly higher in M. croceipes (females: 219–222; males: 220–224) than in C. marginiventris (females: 192–194; males: 193–196). A comparison of males and females within each species demonstrated a striking sexual difference in terms of an enlarged glomerulus (macroglomerulus or MG) at the entrance of the antennal nerve and of a complex of 3–4 MG (CMG) in the posterior region of the antennal lobe of males of both species. Being specific to males, both the MG and CMG might be involved in the detection of female-related odor.     

Sexual dimorphism in the antennal lobe of the ant Camponotus japonicus

The carpenter ant, a social hymenopteran, has a highly elaborated antennal chemosensory system that is used for chemical communication in social life. The glomeruli in the antennal lobe are the first relay stations where sensory neurons synapse onto interneurons. The system is functionally and structurally similar to the olfactory bulbs of vertebrates. Using three-dimensional reconstruction of glomeruli and subsequent morphometric analyses, we found sexual dimorphism of the antennal lobe glomeruli in carpenter ants, Camponotus japonicus. Female workers and unmated queens had about 430 glomeruli, the highest number reported so far in ants. Males had a sexually dimorphic macroglomerulus and about 215 ordinary glomeruli. This appeared to result from a greatly reduced number of glomeruli in the postero-medial region of the antennal lobe compared with that in females. On the other hand, sexually isomorphic glomeruli were identifiable in the dorsal region of the antennal lobe. For example, large, uniquely shaped glomeruli located at the dorso-central margin of the antennal lobe were detected in all society members. The great sexual dimorphism seen in the ordinary glomeruli of the antennal lobe may reflect gender-specific tasks in chemical communications rather than different reproductive roles.     

Caste-specific postembryonic development of primary and secondary olfactory centers in the female honeybee brain

Eusocial insects are characterized by division of labor among a sterile worker caste and a reproductive queen. In the honeybee both female castes are determined postembryonically by environmental factors, and queens develop substantially faster than workers. Since olfaction plays a crucial role in organizing honeybee behavior and social interactions, we compared the development of primary and secondary olfactory centers in the brain. Age-synchronized queen and worker pupae were raised in incubators at 34.5 degrees C, and their external morphology was characterized for all pupal stages. The development of olfactory synaptic neuropil was analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and confocal microscopy. In the antennal lobes of queens olfactory glomeruli formed approximately 4 days earlier than in workers. The adult number of olfactory glomeruli was in a similar range, but the total glomerular volume was slightly smaller in queens. Olfactory and visual subdivisions (lip, collar) of the mushroom-body calyx formed early, whereas the basal ring separated late. Synaptic microglomeruli in the olfactory lip were established approximately 3-4 days earlier in queens compared to workers. We propose that developmental heterochrony results in fewer synapses in olfactory centers (smaller glomeruli, fewer microglomeruli) in queens, which may result in poorer performance on olfactory learning tasks compared to workers.     

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.     

The antennal lobe of the African malaria mosquito, Anopheles gambiae - innervation and three-dimensional reconstruction

Antibody labelling and subsequent three-dimensional reconstructions of the primary olfactory centres, the antennal lobes, of male and female African malaria mosquitoes, Anopheles gambiae, revealed 61 and 60 glomerular neuropils respectively. In addition to the small difference in number of glomeruli, sexual dimorphism was observed in both the size of the antennal lobe and of individual glomeruli. Furthermore, sexual specificity was observed within the array. Anterograde staining of afferents from peripheral olfactory organs support the reconstruction of the glomerular array. Although anterograde stainings support an organotopic organization of the antennal lobe, convergence of afferents originating from different organs into single glomeruli is observed. This finding, in both A. gambiae and A. aegypti, may shed new light upon the development and function of the olfactory system.     

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

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

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

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

Dual olfactory pathway in Hymenoptera: evolutionary insights from comparative studies

In the honeybee (Apis mellifera) and carpenter ant (Camponotus floridanus) the antennal lobe output is connected to higher brain centers by a dual olfactory pathway. Two major sets of uniglomerular projection neurons innervate glomeruli from two antennal-lobe hemispheres and project via a medial and a lateral antennal-lobe protocerebral tract in opposite sequence to the mushroom bodies and lateral horn. Comparison across insects suggests that the lateral projection neuron tract represents a special feature of Hymenoptera. We hypothesize that this promotes advanced olfactory processing associated with chemical communication, orientation and social interactions. To test whether a dual olfactory pathway is restricted to social Hymenoptera, we labeled the antennal lobe output tracts in selected species using fluorescent tracing and confocal imaging. Our results show that a dual pathway from the antennal lobe to the mushroom bodies is present in social bees, basal and advanced ants, solitary wasps, and in one of two investigated species of sawflies. This indicates that a dual olfactory pathway is not restricted to social species and may have evolved in basal Hymenoptera. We suggest that associated advances in olfactory processing represent a preadaptation for life styles with high demands on olfactory discrimination like parasitoism, central place foraging, and sociality.     

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.     

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.     

Spatial representation of odours in the antennal lobe of the moth Spodoptera littoralis (Lepidoptera: Noctuidae)

Glomeruli within the antennal lobe (AL) of moths are convergence sites for a large number of olfactory receptor neurons (ORNs). The ORNs target single glomeruli. In the male-specific cluster of glomeruli, the macroglomerular complex (MGC), the input is chemotypic in that each glomerulus of the MGC receives information about a specific component of the conspecific female sex pheromone. Little is known about how neurons that detect other odorants arborize in and amongst glomeruli. The present study focuses on how sex pheromones and biologically relevant semiochemicals are represented in the ALs of both sexes of the moth Spodoptera littoralis. To assess this, we optically measured odour-evoked changes of calcium concentration in the ALs. Foci of calcium increase corresponded in size and shape with anatomical glomeruli. More than one glomerulus was normally activated by a specific non-pheromonal odorant and the same glomerulus was activated by several odorants. All odorants and pheromone components tested evoked unique patterns of glomerular activity that were highly reproducible at repeated stimulations within an individual. Odour-evoked patterns were similar between individuals for a given odorant, implicating a spatial olfactory code. In addition, we demonstrated that activity patterns evoked by host-plant related volatiles are similar between males and females.     

Three-dimensional organization of the glomeruli in the antennal lobe of the parasitoid wasps <Emphasis Type="Italic">Cotesia glomerata</Emphasis> and <Emphasis Type="Italic">C. rubecula</Emphasis>

Two closely related parasitoid wasp species, Cotesia glomerata and C. rubecula, differ in their use of associative learning. To investigate the neural basis underlying these differences, it is necessary to describe the olfactory pathway of both wasp species. This paper focuses on the organization of the glomeruli in the antennal lobe. Glomeruli were stained by retrograde axon tracing of all axons in the antennal nerve and observed by confocal laser scanning microscopy. Stacks of optical sections were processed with AMIRA software, and 3D digital models of the glomeruli were produced. The combined use of 2D images and 3D surface models of the antennal lobes enabled the identification of a set of corresponding glomeruli in both wasp species. This offers unique opportunities for the study of subtle differences involved in synaptic plasticity that may occur at the glomerular level and factors regulating this plasticity.     

Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli

Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons. © 1995 John Wiley & Sons, Inc.     

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.     

Metamorphic development of locusta-tachykinin immunoreactive neurons of the antennal lobes of the beetle Tenebrio molitor and the effect of fenvalerate application

The effects of the widely used neurotoxic pyrethroid insecticides on neuronal development or plasticity are unclear. To expand knowledge about the influence of the pyrethroid fenvalerate on neuronal development, metamorphic remodelling of the primary olfactory neuropil of the beetle Tenebrio molitor has been studied. The antennal lobe is subdivided into distinct glomeruli before metamorphosis. This is in contrast to that which occurs in other well-studied holometabolous insects such as the moth Manduca sexta and the honeybee. As an indicator of antennal lobe interneurons, locusta-tachykinin immunoreactive neurons have been used. They project into the antennal lobes and form tufted arbors in larval and adult stages within glomeruli throughout the neuropil. These glomerular structures are invaded by glomerular sensory afferent axons and are surrounded by processes of glia cells. With pupation, the glomerulization is lost and no locusta-tachykinin or substance P immunoreactivity is visible in the antennal lobe. The immunoreactivity reappears during metamorphosis, starting with diffusely branched arbors that later become tufted. Application of the neurotoxic insecticide fenvalerate at pupation in sublethal concentrations resulted in a loss or reduction of glomerular pattern formation by neurons and glia cells during metamorphosis. Labelling of antennal sensory axons revealed that the olfactory neuropil was not deafferented, and also that the sensory axons were not organized into a normal glomerular pattern. In addition to the morphological differences, fenvalerate treatment caused locusta-tachykinin immunoreactivity to reappear prematurely during metamorphosis. Possible reasons for fenvalerate-induced alterations in antennal lobe development and their implications for normal development are discussed.