The Journal of Experimental Biology 200, 1873Ð1879 (1997) 1873 Printed in Great Britain © The Company of Biologists Limited 1997 JEB0905

CENTRAL PROJECTIONS OF THE MAXILLARY AND ANTENNAL NERVES IN THE MOSQUITO AEDES AEGYPTI

PAUL G. DISTLER* AND JÜRGEN BOECKH Institut für Zoologie, Universität Regensburg, D-93040 Regensburg, Germany

Accepted 23 April 1997

Summary

In the mosquito Aedes aegypti, CO2-sensitive receptor ipsilateral antennal lobe. There, they terminate within neurones are located together with two other types of an identified glomerulus of the ventroposterior lobe. chemoreceptor neurones in club-shaped sensilla basiconica (2) Afferents of the antennal flagellum project into all on the most distal segment of the maxillary palps. In order glomeruli of the ipsilateral antennal lobe, with the to identify the central target neuropiles of these neurones exception of the glomerulus innervated by the maxillary and to determine whether antennal receptor neurones nerve. The present anatomical findings suggest that project into the same area, the palpal and antennal nerves primary processing of information about CO2 levels takes were labelled by anterograde staining with horseradish place in a defined glomerulus which also receives input peroxidase and by experimentally induced degeneration. from other palpal chemoreceptor neurones. The different methods revealed a consistent projection pattern. (1) Maxillary afferents project into the Key words: insect, olfactory system, carbon dioxide, palpal suboesophageal ganglion and ascend further into the projections, mosquito, Aedes aegypti.

Introduction Mosquitoes, like a number of other blood-sucking insects, are lobe (Anton, 1996). Basically similar projection patterns exist equipped with sensory cells responding to host emanations such in other insects. Both physiological and anatomical evidence for as moisture, heat and, of particular importance, body odours. a single and specifically ‘reserved’ glomerulus in the antennal Among the latter, lactic acid and carbon dioxide have been lobe for CO2 input from the labial palps has been reported in shown to alarm and/or attract female mosquitoes (e.g. Daykin an arctiid moth, Rhodogastria luteibarba (Bogner et al. 1986), et al. 1965; Mayer and James, 1969; Smith et al. 1969; Friend and in two other lepidopteran species, Manduca sexta (Kent et and Smith, 1977; Galun, 1977; Gillies, 1980; for reviews, see al. 1986) and Pieris rapae (Lee and Altner, 1986). Bowen, 1991; Takken, 1991; for more recent results, see Geier, Our interest in the neuronal processing of host odorants in 1995; Steib, 1995). Behavioural and electrophysiological mosquitoes has led us to the question of the location of CO2- studies have shown that the yellow fever mosquito Aedes elicited activity in the brain of A. aegypti. As a first step in aegypti (Diptera: Culicidae) has receptor organs that detect very answering this question, we have studied the afferent low concentrations and also extremely small changes in carbon projections of the maxillary and antennal nerves using two dioxide (CO2) concentration in air (Kellogg, 1970; Grant et al. different labelling techniques: (1) anterograde horseradish 1995; Geier, 1995; Steib, 1995). The CO2-detecting primary peroxidase (HRP) labelling and (2) experimentally induced receptor neurones are located on the most distal, fourth segment degeneration. In the second step, nerve cell activity elicited by of the maxillary palps (Fig. 1). Fine-structure and CO2 stimulation in the central nervous system is located using electrophysiological studies have shown that these neurones lie 2-deoxyglucose labelling (B. Bausenwein and J. Boeckh, in triads together with other types of chemosensory receptor unpublished) in order to identify further the pathway for the cells within club-shaped sensilla basiconica (also referred to as processing of this important olfactory input. palpal pegs). Approximately 30 such sensilla are found on a single maxillary palp of a female A. aegypti (McIver and Materials and methods Charlton, 1970; Chaika, 1977; McIver, 1982). More recently, tracing of the axonal projections of these neurones with Animals fluorescent dye has revealed their projections in a very distinct Adult 1- to 2-week-old females of the yellow fever mosquito glomerulus-like terminal region within the ipsilateral antennal Aedes aegypti L. (Diptera: Culicidae) were used as

*e-mail: [email protected]. 1874 P. G. DISTLER AND J. BOECKH experimental animals. They were taken from a colony reared fixed in OsO4 and embedded in resin (see below). HRP- in the Institute’s vivarium under standard conditions (Geier, labelled brains were embedded in a mixture of 1995). ovalbumin/gelatin, fixed for a further 10 min, and transferred into 0.01 mol l−1 PB, pH 7.4. Anterograde tracing of the maxillary and antennal nerves HRP labelling Histochemical reaction of HRP The animals were immobilized by exposure to cold air (8 ¡C) HRP-labelled brains were cut into 30 µm thick serial and subsequently fixed in a position favourable for sections in a horizontal plane using a vibroslicer. The sections − manipulations on the maxillary palps. In 20 mosquitoes, the were collected in 0.05 mol l 1 TrisÐHCl buffer (pH 7.5). HRP fourth segment of one maxillary palp was cut off just distal to was visualized by reaction with nickel-intensified its base using a fine blade. A small drop of a 5 % aqueous diaminobenzidine (Ni-DAB) (0.02 % DAB in TrisÐHCl buffer, solution of HRP (Boehringer, Mannheim, Germany) was pH 7.5, with 0.1 % nickel ammonium sulphate and 0.003 % immediately placed onto the lesion and allowed to infiltrate for hydrogen peroxide). The reaction was monitored under the a few seconds. Finally, the preparation was sealed with Vaseline. microscope and stopped after 5 min by removing the Ni-DAB A similar labelling procedure had been used previously to trace solution. The sections were postfixed in 1 % OsO4 dissolved in the central projections of the cockroach antennal nerve (Kirn, distilled water for 20 min at RT and, after dehydration in 1996). To label the antennal nerve with HRP, the flagellum was ethanol, embedded in Epon 812. The polymerization cut off just distal to the pedicel in 14 specimen, and HRP was temperature was 57 ¡C. applied onto the lesioned site as described above. The animals were kept in a moist chamber for between 2.5 and 8 h at room Semi- and ultrathin sectioning temperature (20 ¡C) before the preparations were fixed. For reconstruction of the nerve projections, 3 µm thick semithin section series were cut from whole brains (induced Induced degeneration degeneration) or from selected 30 µm thick sections (HRP Degeneration studies (see Boeckh et al. 1970) of both the labelling) and counterstained with Richardson Blue. For maxillary and the antennal nerve projections were performed maxillary nerve labelling, three brains with induced degeneration in four specimens each. The fourth segment of the maxillary (24 and 48 h) and five brains with HRP labelling (between 5 and palp or the flagellum, respectively, was removed according to 8 h) were studied. For antennal nerve labelling, three brains with the procedure described above, in order to induce degeneration induced degeneration (24 and 48 h) and six brains with HRP of receptor axons. These preparations were fixed 24 or 48 h labelling (between 5 and 8 h) were studied. In addition, ultrathin post lesion. During the degeneration period, the animals could sections were cut from (a) the ventroposterior glomerulus with move freely. the terminal arborizations of the maxillary afferents (three different preparations) and (b) the nerve bundle in the Fixation of the brains suboesophageal ganglion (one preparation). These sections were After decapitation of the mosquitoes, the head capsules were counterstained with uranyl acetate (20 min) and lead citrate fixed using rapid-setting glue on their posterior surface and (3 min), and examined in a Zeiss electron microscope (EM 109). superfused with 2.5 % glutaraldehyde in 0.1 mol l−1 phosphate buffer (PB), pH 7.4. In order to achieve immediate fixation of the brain, the head capsules were carefully opened by ablating Results small areas of cuticle in the dorsal region using sharp glass Anterograde tract tracing of the maxillary palp nerve capillary tips. The free-floating heads were fixed for HRP labelling of maxillary afferents revealed a distinct fibre approximately 1Ð1.5 h at room temperature (RT). Preparations bundle that projected into the suboesophageal ganglion and that had been induced to degenerate were subsequently post- ascended further into the ipsilateral antennal lobe. There, all

Fig. 1. Scanning electron micrographs of the head and mouthparts of a female Aedes aegypti (photographs kindly provided by A. Kühn). (A) Head with antenna (an) and maxillary palps (mp). (B) Distal (fourth) segment of the maxillary palp showing the sensilla basiconica (arrow) and non- innervated hairs (arrowhead). (C) Single basiconic sensillum (arrow) at higher magnification. Scale bars, A, 200 µm; B, 50 µm; C, 10 µm. Maxillary and antennal nerve projections 1875

projected over a short distance into the contralateral neuropile. Experimentally induced degeneration of the maxillary palp nerve for 24 and 48 h revealed a similar projection pattern (Fig. 2B). The characteristic degeneration granules were found in the same glomerulus that had been identified by HRP labelling. Fine-structure analysis showed that the glomerulus was demarcated within the surrounding neuropile by a thin glial sheath (Fig. 3). Only laterally, next to the basal antenno- cerebral tract, was the glial border not clearly identifiable. The HRP-labelled axon processes were distributed throughout the glomerulus neuropile. A subdivision of this into regions with either dense or less-dense innervation was not evident. By analogy with the light microscopic findings, the corresponding contralateral glomerulus revealed no (two brains) or only very few degenerated processes (one brain). Cross sections through the maxillary nerve in the suboesophageal ganglion revealed that the majority of fibres were HRP-labelled (Figs 4, 5). The corresponding unlabelled maxillary nerve in the contralateral hemibrain contained approximately 110, mostly small, axons.

Anterograde tract tracing of the antennal nerve HRP labelling of the antennal (flagellar) axons revealed that such afferents innervated all the antennal lobe glomeruli of the ipsilateral hemibrain, with the exception of the ventroposterior glomerulus that had specifically received the maxillary afferents (Fig. 6A,B). Contralateral projections were not evident, even after HRP labelling for up to 8 h (14 brains). Slight differences were found, however, between different Fig. 2. Maxillary nerve afferents. (A) 30 µm thick horizontal section preparations with regard to both the number and the intensity through a brain with horseradish-peroxidase (HRP)-labelled maxillary of labelled receptor cell axons. Induced degeneration of the afferents (right maxillary nerve labelled, 5.5 h). A densely innervated antennal nerve for periods of 24 and 48 h (three brains) glomerulus (g) is evident in the ipsilateral ventroposterior region of revealed a similar projection pattern (Fig. 6C). the antennal lobe (AL). No labelled fibre processes are present in the corresponding contralateral brain region. The innervated glomerulus lies lateral to the basal region of the antenno-cerebral tract (act) Discussion and medial to the oesophagus. For further details, see Fig. 3. (B) Richardson-Blue-stained 3 µm thick horizontal section through a Morphology of maxillary and antennal afferents brain with experimentally induced degeneration of maxillary afferents Both HRP labelling and induced degeneration of the (24 h). Degeneration granules are present in only one ventroposterior maxillary afferents consistently revealed projections into a glomerulus (g). Note that the corresponding contralateral glomerulus single glomerulus located in the ipsilateral ventroposterior (asterisk) is not innervated. The spatial positions of both glomeruli antennal lobe (Figs 2Ð4). Its contralateral counterpart did not identified by HRP labelling and by induced degeneration are identical. contain labelled processes, except for a very few in one act, antenno-cerebral tract; cb, central body; PC, protocerebrum. Scale specimen. These presumably belonged to receptor cells of the bars, 50 µm. contralateral maxillary palp, which could have been severed during the operation at the closely neighbouring palp and labelled axons terminated within a single, oval-shaped and thereby been caused to degenerate. Preparations labelled for particularly large glomerulus approximately 30Ð50 µm in varying periods, from 2.5 to 48 h, did not show significant diameter (Figs 2–5). This projection pattern was identified in differences in the projection pattern and the terminal regions 10 brains. No morphological differences with regard to the of the axonal processes, indicating that the nerve had been projection site or the number of innervated glomeruli were completely labelled. This was further supported by the finding evident in brains stained for between 2.5 and 8 h. The that all axons within the maxillary nerve appeared to be innervated glomerulus was located in the ventroposterior labelled. Earlier morphological and fine-structure antennal lobe. Medially, it bordered the oesophagus and investigations on the maxillary receptor cell types of the fourth laterally it bordered the root of the antenno-cerebral tract palpal segment had described a similar number of receptor (Fig. 2A). No axonal projections were detected within the cells (McIver, 1982). The ventroposterior glomerulus contralateral antennal lobe. In the suboesophageal ganglion, identified here is located in the same antennal lobe region as however, a few smaller processes crossed the midline and the axon terminals found in the fluorescent dye labellings of 1876 P. G. DISTLER AND J. BOECKH

Fig. 3. Ultrathin section through the ventroposterior glomerulus innervated by HRP-labelled maxillary afferents (same section as in Fig. 2A). Stained processes (arrows) are distributed throughout the glomerulus. act, basal region of the antenno-cerebral tract; AL, antennal lobe neuropile; G, ventroposterior glomerulus. Scale bar, 10 µm.

Anton (1996). A slight difference between the studies concerns The maxillary projection pattern in A. aegypti shows certain the number of innervated glomeruli. In the present study, parallels with the labial afferents in some lepidopteran species. terminal ramifications of afferent fibres were found only in one Bilateral axonal projections from each labial palp have been large glomerulus (Figs 2–4), and the fluorescence appeared to shown to terminate mirror-symmetrically in a single be distributed over two smaller, very closely neighbouring glomerulus in each antennal lobe in R. luteibarba (Bogner et glomeruli. In contrast to other cyclorrhaphic Diptera (Boeckh et al. 1970; Stocker et al. 1990; Stocker, 1994), the maxillary afferents projected exclusively into the ipsilateral antennal lobes. Only in the suboesophageal ganglion were a few fibres found to cross the midline. The majority of the maxillary afferents identified in Drosophila melanogaster bifurcate and project into both antennal lobes. There, they terminate within a small, mirror-symmetrical group of three (five) ventroposterior glomeruli (Stocker et al. 1990; Singh and Nayak, 1985). Morphologically, the maxillary afferents in D. melanogaster and A. aegypti differ with regard to (a) the projection site and (b) the number of antennal lobe glomeruli innervated. Such differences might be due, for example, to different numbers of chemoreceptor cells on the palps in the two species (for a review, see Stocker, 1994). In Fig. 4. HRP-labelled right maxillary nerve in the suboesophageal hemimetabolous insects (Locusta migratoria, Ernst et al. 1977; ganglion (sog) (same brain as in Fig. 2A). The fibres (arrow) project Altman and Kien, 1986; Periplaneta americana, Boeckh and towards the centre of the neuropile, where small ramifications are Ernst, 1987), the maxillary nerve projects into both the ipsi- evident (arrowhead). Few processes cross the midline (none shown and the contralateral brain hemisphere. There, the terminal here). 30 µm thick horizontal section. og, optic ganglia. Scale bar, ramifications are located in the so-called lobus glomeratus. 100 µm. Maxillary and antennal nerve projections 1877

Fig. 5. Ultrathin section through the maxillary nerve in the suboesophageal ganglion. (A) HRP-labelled nerve. (B) Maxillary nerve (unlabelled) in the contralateral hemiganglion. Arrows, axonal processes of labelled (A) and unlabelled (B) receptor neurones. Scale bar, 1 µm. al. 1986), P. rapae (Lee and Altner, 1986) and M. sexta (Kent et al. 1986). In Rhodogastria, furthermore, it has been demonstrated that the respective receptor cells in the distal labial pits are very sensitive to CO2 and only slightly sensitive to other odorants (Bogner, 1990). The CO2-sensitive receptor neurones constitute the majority of labial afferents in Rhodogastria. Anterograde tracing of the antennal nerve in A. aegypti revealed projections only into the ipsilateral antennal lobe. All the glomeruli, with the exception of the ‘maxillary’ glomerulus, were innervated (Fig. 6). These findings are in accordance with data reported in a previous fluorescence labelling study (Anton, 1996). Ipsilateral projections of antennal chemoreceptor cells are typical of many insect species (for reviews, see Boeckh and Tolbert, 1993; Hildebrand, 1996). In cyclorrhaphic Diptera such as Musca and Drosophila, however, the majority of antennal chemoreceptors have axon projections into both the ipsi- and the contralateral antennal lobe (Boeckh et al. 1970; Stocker 1994). In D. melanogaster, it has been reported that glomeruli innervated by the maxillary afferents receive no other antennal afferents (Singh and Nayak, 1985; Stocker et al. 1990). These findings are consistent with the situation in A. aegypti, where no antennal axons terminated in the ventroposterior glomerulus. Studies on the central

Fig. 6. Antennal nerve afferents. (A) 30 µm thick horizontal section (overview) through a brain with HRP-labelled antennal afferents (right antennal nerve stained, 5 h). (B) All glomeruli of the ipsilateral antennal lobe, with the exception of the ventroposterior glomerulus, are innervated. (C) Induced degeneration (24 h) of the right antennal nerve reveals a similar innervation pattern. (B,C) 3 µm thick horizontal sections counterstained with Richardson Blue. Arrows, innervated glomeruli; asterisks, ventroposterior glomeruli. act, antenno-cerebral tract; AL, antennal lobe; cb, central body; PC, protocerebrum. Scale bars, A, 100 µm; B,C, 50 µm. 1878 P. G. DISTLER AND J. BOECKH projections of the antennal nerve in Lepidoptera (M. sexta, References Kent et al. 1986; P. rapae, Lee and Altner, 1986; ALTMAN, J. S. AND KIEN, J. (1986). Functional organization of the Rhodogastria, Bogner et al. 1986) also revealed innervation of subesophageal ganglion in arthropods. In Arthropod Brain: Its all antennal lobe glomeruli with the exception of the one Evolution, Development, Structure and Functions (ed. A. P. Gupta), receiving the labial afferents. pp. 265Ð302. New York: Wiley. ANTON, S. (1996). Central olfactory pathways in mosquitoes and other Functional considerations insects. In Proceedings of the Ciba Foundation, Symposium no. In the present study, all the labelled maxillary afferents 200, Olfaction in MosquitoÐHost Interactions, pp. 184Ð196. Chichester: Wiley. terminated within a single glomerulus, suggesting that the BOECKH, J., DISTLER, P., ERNST, K. D., HÖSL M. AND MALUN, D. latter might be a distinct region of the antennal lobe specialized (1990). Olfactory bulb and antennal lobe. In Chemosensory for the primary processing of CO2. The hypothesis of a ‘CO2- Information Processing (ed. D. Schild), pp. 201Ð228. Berlin: specific’ glomerulus is supported by the observation that in A. 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Verhaltensversuche mit Gelbfiebermücken Aedes The present findings show that the glomerulus described aegypti zur Aufklärung des attraktiven Reizmusters bei der here in A. aegypti represents probably the only and specific olfaktorischen Wirtsfindung. PhD thesis, Universität Regensburg. target area for CO2-sensitive afferents, which is analogous to GILLIES, M. T. (1980). The role of carbon dioxide in host-finding by the situation in R. luteibarba. However, there is combined mosquitoes (Diptera: Culicidae): a review. Bull. ent. Res. 70, anatomical and physiological evidence for other 525Ð532. chemoreceptor afferents in this glomerulus, suggesting that, in GRANT, A. J., WIGTON, B. E., AGHAJANIAN, J. G. AND O’CONNEL, R. J. (1995). Electrophysiological responses of receptor neurons in contrast to the situation in R. luteibarba, further odorants are mosquito maxillary palp sensilla to carbon dioxide. J. comp. probably processed in this neuropile in A. aegypti. Physiol. A 177, 389Ð396. 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