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

https://doi.org/10.1016/j.asd.2006.06.004Get rights and content

Abstract

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.

Introduction

Primary olfactory centres, like antennal lobes (ALs) of insects and olfactory bulbs of vertebrates, are characterised by their subdivision into structural units called glomeruli (reviewed by Anton and Homberg, 1999). Within the glomerular neuropil, synaptic integration between olfactory afferents and dendritic arborisations of their target interneurons has been postulated to underlie the complex mechanisms of odour identification and discrimination (reviewed by Christensen and White, 2000, Hansson and Christensen, 1999, Ignell and Hansson, 2005). As such, the glomerular array is thought to constitute a chemotopic map, which ultimately forms the basis of an olfactory code (reviewed by Christensen and White, 2000, Galizia and Menzel, 2000, Galizia and Menzel, 2001, Ignell and Hansson, 2005). Detailed analysis of glomerular arrays has, so far, only been reported from insects. These analyses generally show a low intraspecific variance in the number, shape, size and position of glomeruli (Arnold et al., 1985, Berg et al., 2002, Chambille and Rospars, 1981, Galizia et al., 1999, Laissue et al., 1999, Rospars, 1983, Rospars and Chambille, 1989, Rospars and Hildebrand, 1992, Schachtner et al., 2005, Smid et al., 2003). However, variation, particularly concerning the number of glomeruli between species, is apparent; most species studied so far have approximately 50 to 160 glomeruli, with a few species having 1000 or more (reviewed by Anton and Homberg, 1999, Schachtner et al., 2005). Although most glomeruli are sexually isomorphic, sexual ‘specificity’ (Rospars and Hildebrand, 2000) within the glomerular array has been reported in moths, cockroaches and bees, where a macroglomerulus or a macroglomerular complex involved in pheromone information processing is clearly identifiable in male individuals (reviewed by Anton and Homberg, 1999). Male-specific enlargement, or sexual dimorphism (Rospars and Hildebrand, 2000), of sexually homologous glomeruli has also been reported (Kondoh et al., 2003).

The organisation of the central olfactory system in mosquitoes has, despite the established role of olfactory cues mediating several behavioural expressions of these species (Bowen, 1991, Clements, 1999, Takken and Knols, 1999), not attracted a general interest. Due to the socio-economic importance of these disease vectors, however, an increased understanding of the chemical ecology of mosquitoes and their neuroethology has recently been recognised to be of key importance for designing future control strategies (Zwiebel and Takken, 2004). As a first step in unravelling the complexity of the olfactory system of mosquitoes we have characterised its neuronal architecture in A. gambiae and in the yellow fever mosquito, Aedes aegypti (Ignell et al., 2005). We have also presented a three-dimensional reconstruction of the AL of male and female A. aegypti, based on high-resolution staining of the glomerular neuropil (Ignell et al., 2005). That study indicated a sexual ‘specificity’ in the topography and number of glomeruli, as well as sexual dimorphism within the glomerular array indicated by differentially enlarged glomeruli in both sexes. This ‘specificity’ and dimorphism likely reflect the divergent behavioural requirements of the sexes. Like A. aegypti, the well characterised anthropophilic and oviposition behaviours of female A. gambiae are elicited by cues not required by males for their general behavior (Takken and Knols, 1999). Furthermore, we identified putative sexually isomorphic glomeruli. These glomeruli are likely to receive convergent input from similarly tuned olfactory receptor neurons (ORNs) and thus be topographically ‘conserved’ neuropil. Both sexes are e.g., dependent on sugar/nectar feeding as a general energy supply, and plant odour information is thus required to be processed in the AL of both (Takken and Knols, 1999). Functional staining studies are, however, required to verify the function of sexually ‘specific’, dimorphic and isomorphic glomeruli.

In the present study, we present a three-dimensional reconstruction of the glomerular organisation of the AL of A. gambiae based on high-resolution staining of the neuropil using monoclonal nc82 antibody staining. An online 3D reconstruction of the antennal lobe is available at http://www.vsv.slu.se/chemosensmosquito/AngambiaeAL. In addition, we present data from anterograde stainings from antennae, maxillary palps and the labium, the peripheral organs that carry olfactory sensilla (McIver, 1982). These stainings allowed us to identify the sensory terminal regions in the AL of the ORNs residing within these sensory units.

Section snippets

Insects

The Anopheles gambiae colony used originated from Suakoko, Liberia (eggs courtesy of Dr. W. Takken). Egg batches were kept in plastic containers (20 × 18 × 7 cm) at 28.5 °C, 80% RH and a L:D cycle of 12:12 h without artificial dusk period. Larvae were fed with Tetramin™ fish food until pupation. Pupae were transferred to plastic cylindrical netted cages (20 × 30 cm) for adults to emerge; males and females were allowed to consort together for mating. Adults were given 6% sugar-water solution.

The antennal lobe neuropil of Anopheles gambiae

In A. gambiae, the ALs protrude ventrally on either side of the oesophagus (Fig. 1). The AL neuropil receives afferent innervation from the antennal flagellum, the maxillary palp (this study; Ignell et al., 2005) as well as the labium (this study). In addition, axons originating from Johnston's organ terminate in a multi-lobed neuropil, Johnston's Organ Center (JOC), which is situated as a wedge in the AL surrounded by glomeruli on all sides except ventrally (Fig. 3, Fig. 5). The neuronal

Histological staining and delineation of glomeruli

nc82 and other monoclonal antibodies like synaptotagmin are currently being used for structural studies of the olfactory neuropil in various species of insects (Huetteroth and Schachtner, 2005, Ignell et al., 2005, Kondoh et al., 2003, Laissue et al., 1999, Vosshall et al., 2000, Wong et al., 2002). As for these studies, application of monoclonal antibody stainings generally provided a more or less distinct staining of all glomeruli in A. gambiae. Certain areas within the glomerular array were,

Acknowledgements

We thank Teun Dekker, Marcus Sjöholm, Marcus Stensmyr, Maryam Ghadimi and Purayil Siju for technical assistance. Furthermore, we would like to thank the two anonymous reviewers for helpful comments.

References (49)

  • N. Thorne et al.

    Taste perception and coding in Drosophila

    Current Biology

    (2004)
  • L. Vosshall et al.

    A spatial map of olfactory receptor expression in the Drosophila antenna

    Cell

    (1999)
  • J.W. Wang et al.

    Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain

    Cell

    (2003)
  • Z. Wang et al.

    Taste representations in Drosophila brain

    Cell

    (2004)
  • A.M. Wong et al.

    Spatial representation of the glomerular map in the Drosophila protocerebrum

    Cell

    (2002)
  • L.J. Zwiebel et al.

    Olfactory regulation of mosquito host interactions

    Insect Biochemistry and Molecular Biology

    (2004)
  • S. Anton et al.

    Antennal lobe structure

  • S. Anton et al.

    Quantitative analysis of olfactory receptor neuron projections in the antennal lobe of the malaria mosquito, Anopheles gambiae

    Journal of Comparative Neurology

    (2004)
  • G. Arnold et al.

    Comparative study of the antennal lobes and their afferent pathway in the worker bee and the drone (Apis mellifera)

    Cell and Tissue Research

    (1985)
  • B.G. Berg et al.

    Digital atlases of the antennal lobe in two species of tobacco budworm moths, the oriental Helicoverpa assulta (Male) and the American Heliothis virescens (Male and Female)

    Journal of Comparative Neurology

    (2002)
  • G. Boyan et al.

    Organization of the commissural fibers in the adult brain of the locust

    Journal of Comparative Neurology

    (1993)
  • K.S. Boo

    Antennal sensory receptors of the male mosquito, Anopheles stephensi

    Zeitschrift für Parasitenkunde

    (1980)
  • M.F. Bowen

    The sensory physiology of host seeking behaviour in mosquitoes

    Annual Review of Entomology

    (1991)
  • T.A. Christensen et al.

    Representation of olfactory information in the brain

  • Cited by (55)

    • First neuronal projection from Haller's organ to the synganglion and three-dimensional reconstruction of Amblyomma sculptum olfactory lobe

      2021, Ticks and Tick-borne Diseases
      Citation Excerpt :

      In the present study, the mean glomerular volumes of the male and the females were similar and rather smaller than those of other hematophagous arthropods (Ghaninia et al., 2007; Ignell et al., 2005; Ye et al., 2020). Females and males of the mosquitoes A. gambiae and A. aegypti present sexually dimorphic glomeruli: this condition is associated with different food preferences, since only the females are hematophagous (Ghaninia et al., 2007; Ignell et al., 2005). Both males and females of A. sculptum are blood feeders, so there are no differences in food preferences (Randolph, 2014).

    • Disruption of Mosquito Olfaction

      2016, Genetic Control of Malaria and Dengue
    • Reception of odors and repellents in mosquitoes

      2015, Current Opinion in Neurobiology
      Citation Excerpt :

      These receptors are probably expressed in similar numbers of classes of odorant receptor neurons (ORNs) housed in sensilla on the antenna, maxillary palps and proboscis. The axons of the ORNs project to the antennal lobe (AL) in the brain's deutocerebrum, where they innervate glomeruli, probably sorting according to their expressed receptors [6–8]. The Or family is the largest, most diverse, with ligands known for a majority of members in Anopheles gambia [9,10].

    • Olfactory disruption: Toward controlling important insect vectors of disease

      2015, Progress in Molecular Biology and Translational Science
      Citation Excerpt :

      As these disease vectors use unique combinations of volatiles to locate a bloodmeal, corresponding anatomical features of their olfactory neuropil provide logical targets for behavioral disruption. No AL macroglomerular complexes have been described and only slight AL sexual dimorphism has been reported for mosquitoes.131,147 It is possible that other features of the mosquito olfactory system such as differences in receptor gene expression account for sexually dimorphic behaviors, e.g., host seeking in blood-feeding females.

    View all citing articles on Scopus
    View full text