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