A Confined Taste Area in a Lepidopteran Brain

Arthropod Structure & Development 35 (2006) 35–45 www.elsevier.com/locate/asd

A conﬁned taste area in a lepidopteran brain

Pa˚l Kvello, Tor J. Almaas, Hanna Mustaparta *

Department of Biology, Norwegian University of Science and Technology, Neuroscience Unit, MTFS, NO 7489 Trondheim, Norway

Received 11 July 2005; accepted 20 October 2005

Abstract

Knowledge about the neuronal pathways of the taste system is interesting both for studying taste coding and appetitive learning of odours. We here present the morphology of the sensilla styloconica on the proboscis of the moth Heliothis virescens and the projections of the associated receptor neurones in the central nervous system. The morphology of the sensilla was studied by light microscopy and by scanning- and transmission electron microscopy. Each sensillum contains three or four sensory neurones; one mechanosensory and two or three chemosensory. The receptor neurones were stained with neurobiotin tracer combined with avidin-ﬂuorescein conjugate, and the projections were viewed in a confocal laser-scanning microscope. The stained axons entered the suboesophageal ganglion via the maxillary nerves and were divided into two categories based on their projection pattern. Category one projected exclusively ipsilaterally in the dorsal suboesophageal ganglion/tritocerebrum and category two projected bilaterally and more ventrally in the suboesophageal ganglion conﬁned to the anterior surface of the neuropil. The bilateral projecting neurones had one additional branch terminating ipsilaterally in the dorsal suboesophageal ganglion/tritocerebrum. A possible segregation of the two categories of projections as taste and mechanosensory is discussed. q 2005 Published by Elsevier Ltd.

Keywords: Heliothis virescens; Proboscis; Sensilla styloconica; Taste neurone projections; Suboesophageal ganglion; Tritocerebrum

1. Introduction allowing non-volatile compounds to enter the lumen and stimulate the receptor neurones. One prominent type of contact The biological importance and early evolutionary origin of chemosensillum in herbivorous lepidopteran species is the the sense of taste is apparent from its ubiquitousness sensillum styloconicum located on the proboscis. As described throughout the animal kingdom. The major role is regulation in Vanessa cardui (Nymphalidae), Choristoneura fumiferana of feeding behaviour like detection and discrimination of food (Clem.) (Tortricidae) and Rhodogastria bubo Walker (Arc- sources and toxic items. In some animals, the sense of taste tiidae) these sensilla contain three or four sensory neurones, of may also be involved in oviposition and pheromone communi- which two or three are gustatory and one is mechanosensory cation, which is well demonstrated in insects (Sta¨dler and (Altner and Altner, 1986; Krenn, 1990; Walters et al., 1998). Roessingh, 1991; Ramaswamy et al., 1992; Bray and Amrein, Electrophysiological recordings have shown that each gusta- 2003). Reflecting the function, the taste organs of insects tory receptor neurone in a contact chemosensillum seems to be (contact chemosensilla) are present on the mouthparts, specified for one taste category including sugars, salts, water, antennae, tarsl, ovipositor and wings (Dethier, 1976; Pollack, deterrents and in some cases amino acids (Hodgson, 1957; 1977; De Jong and Sta¨dler, 2001). They generally appear as Wolbarsht and Dethier, 1958; Evans and Mellon, 1962; hair-formed structures comprising a thick cuticle wall sur- Shiraishi and Kuwabara, 1970; Dethier, 1976; Liscia and rounding an inner lumen with the dendrites of two to four Solari, 2000; Chyb et al., 2003; Thorne et al., 2004). gustatory neurones (Altner and Altner, 1986; Krenn, 1990; Revealing the neuronal pathways of the taste system is Singh, 1997; Pollack and Balakrishnan, 1997; Walters et al., important for studying how gustatory information is coded in 1998). A mechanosensory neurone is usually attached to the the central nervous system, leading to the different responses of feeding behaviour. The taste pathways are also interesting with base of the hair. The apical part of the cuticle is perforated respect to their role in appetitive learning where sucrose is used as the unconditioned stimulus (Hammer and Menzel, 1995; * Corresponding author. Tel.: C47 73596268; fax: C47 73598294. Hartlieb, 1996; Skiri et al., 2005). To understand the neural E-mail address: [email protected] (H. Mustaparta). mechanisms of appetitive learning it is important to trace the 1467-8039/$ - see front matter q 2005 Published by Elsevier Ltd. involved neural pathways (taste and olfaction) and reveal their doi:10.1016/j.asd.2005.10.003 neuronal connection. Whereas the olfactory pathways have 36 P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45 been extensively studied in several insect species (Hildebrand emerging the pupae were separated according to sex and put and Montague, 1986; Menzel et al., 2005; Stocker, 2001; in a glass container (hight: 18 cm, width: 12 cm, depth: 17 cm) Mustaparta, 2002), the taste pathways are less explored. In the covered by a perforated plexiglass. The container with pupae honey bee Apis mellifera as well as in the flies Phormia regina was kept in a Refritherm 6 E incubator (Struers) at a reversed and Drosophila the axonal projections from bimodal contact photoperiod (14 h light and 10 h dark) and at a temperature of chemosensilla on the external mouthparts have been traced to 22–23 8C. When emerged, the adults were placed into a the suboesophageal ganglion (SOG) (Rehder, 1989; Murphey plexiglass cylinder (height: 20 cm, diameter: 10 cm) covered et al., 1989; Edgecomb and Murdock, 1992). In the flies the by a perforated lid. The moths could feed ad. lib. on a 0.15 M taste neurones seem to project in a different region than sucrose solution. The moths were one to three days old when the associated mechanosensory neurones, and in Drosophila used in the experiments. the phagostimulant (trehalose) neurones project in a different area than the deterrent (caffeine) neurones (Thorne et al., 2004; 2.2. Electron microscopy Wang et al., 2004). An additional projection area is shown for the sensory neurones associated with the pharyngeal contact The outer morphology of the s. styloconica were examined chemosensilla of Drosophila (Stocker, 1994). These neurones with a scanning electron microscope (SEM) at 15–25 kV project in the tritocerebrum which is located anterior-dorsally (JEOL JSM-25S). The proboscis of two individuals were fixed to the SOG and are connected to the labro-frontal nerve in a buffered 0.135% glutaraldehyd-3.6% formaldehyd solution (Strausfeld, 1976). The anatomical border between these two (0.1 M phosphate buffer, pH 7.4) for 3 days at 4 8C dehydrated areas (SOG and tritocerebrum) is indefinable in higher insects, in ethanol and left to air dry for 1 day. The material was then but they are still ascribed to specific behavioural tasks mounted onto SEM viewing aluminium stubs using a carbon (Chaudonneret, 1987; Altman and Kien, 1987; Rajashekhar adhesive tape. The carbon tape was further connected to the and Singh, 1994b). One octopaminerge neurone connecting the aluminium stubs by carbon pasta. The material was coated with taste- and the olfactory system has been identified in the honey a 30 nm thick layer of gold–palladium (JOEL FINE COAT ion bee as the ventral unpaired medial neurone (VUM-mx1) which sputter JFC-1100) before examination in SEM. plays a major role in appetitive learning in this species The inner morphology of the s. styloconica on the proboscis (Hammer, 1993). This interneurone, with dendritic-axonal was examined with a transmission electron microscope (TEM) arborisations in the olfactory neuropils, responds to sucrose, it at 60 kV (JEOL JEM-1200). The proboscides of two has the soma in the ventral SOG and dendritic arborisations in individuals were cut at the base and immediately fixed in the dorsal SOG and tritocerebrum. buffered 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.4) In the noctuid moth Heliothis virescens, the interest is to over night at 4 8C. The material was subsequently postfixed in reveal the physiological properties and the anatomical path- buffered 1% osmium tetroxide (OsO4) for 1.5 h at room ways involved in contact chemosensation as well as the temperature, dehydrated in ethanol and embedded in a medium neuronal connections between this pathway and the olfactory mixture of Durcupan (Fluka) through progressive series of centres. The olfactory system is well studied as concerns the durcupan/propylene oxide (1/5, 1/3, 1/1 and 1/0). By the use of receptor neurone specificity and the central pathways (Almaas an ultratome (ULTRACUT, Reichert-Jung) equipped with a and Mustaparta, 1991; Berg et al., 1998; Stranden et al., 2003; diamond knife (Diatome), serial ultrathin sections (70 nm) Skiri et al., 2004; Røstelien et al., 2005; Mustaparta and were made and collected on one-hole grids (Tebra 1GC Stranden, 2005). Olfactory learning of relevant plant odours is 12H/10H) covered with a pioloform membrane (Pioloform, also demonstrated in this species by the use of the proboscis Agar Scientific LTD). Finally the sections were stained with extension reflex (Skiri et al., 2005). Electrophysiological uranyl acetate for 30 min and rinsed in distilled water before recordings from the contact chemosensilla on the proboscis being stained with lead citrate for 5 min (standard procedure). have been performed showing responses to various phagostimulants and potential deterrents (Blaney and Simmonds, 2.3. Staining of the receptor neurones 1988). The morphology of these sensilla has not been studied, and the projections of taste neurones are not known in any adult The insects were mounted in a plastic tube and wax was lepidopteran species. The present study describes the mor- used for further immobilization of the head and the mouthparts. phology of the s. styloconica on the proboscis of H. virescens The proboscis was uncoiled, the two galeae separated and and the axonal projections of the associated sensory neurones fastened to the wax with tungsten cramps. The distal part of the in the SOG and tritocerebrum. The axons and the target areas two galeae was covered with distilled water and the proximal are shown in three-dimensional reconstructions. part was covered with vaseline to avoid drinking. In 15 preparations one to five s. styloconica were cut. This was 2. Materials and methods carried out under a drop of distilled water and left for 10 min for osmotic distension of the cut dendrites. A few crystals of 2.1. The animals neurobiotin tracer (SP-1120, Vector Labortories, Inc.) were subsequently dissolved in the water. The preparation was The moths were imported as pupae from a laboratory culture placed in a petri dish moistened with a wet piece of paper. The at Novartis Crop Protection, Basel, Switzerland. Before petri dish was placed in the refrigerator for 7 days at 4 8Cto P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45 37

Fig. 1. Morphology of the s. styloconica on the proboscis of H. virescens. Light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). (A) Distal part of the proboscis showing both galeae with the distribution of s. styloconica (Ss) (LM). (B) Distal part of the left galea showing the arrangement of the s. styloconica. Note that the s. styloconica are located on the same area of the proboscis as the drinking slits made up between the dorsal galeal linking structures (DGLS) (SEM). (C) A single s. styloconicum. The outer structure is characterised by a proximal stylus (St) with six ridges (R) and a distal cone (C) (SEM). (D and E) The cone has a distinct socket (Cs) (note the thin cuticle) (SEM and TEM). (F and G) The cone has a single pore (P) at the tip with a diameter of approximately 200 nm (SEM and TEM). Pt proboscis tip. allow diffusion of the dye. The moth was then mounted on a with a centrally located indentation filled with plexiglass-holder for brain dissection. The brains were fixed in methylsalicylate. 4% paraformaldehyde (4 h in room temperature or over night at 4 8C) and rinsed in 0.1 M phosphate buffer (three times during 2.4. Confocal laser-scanning microscopy 30 min) before being placed in 0.1 M phosphate buffer containing 0.3% triton X (six times during 6 h at room The projections of stained receptor neurones in the moth temperature). The brains were then incubated over night at 4 8C brain were examined with a confocal laser-scanning micro- in a 0.1 M phosphate buffer containing 0.05% triton X and the scope (CLSM) (LSM-510, Carl Zeiss), using dry objectives avidin-fluorescein conjugate (A-821, molecular probes). (plan-Neofluar). The green fluorescent dye fluorescein was Subsequently they were rinsed in phosphate buffer (three exited by a 488 nm argon laser at intensity of 6–10 mW and times during 30 min) before dehydrated in ethanol (50, 70, 90, filtered through a bandpass filter (BP 500-550 IR). The brains 96 and 100%). Finally the brains were cleared in methylsali- were scanned in frontal planes with an average of four scans per cylate and oriented in a frontal position on aluminium slides slice to reduce random noise. The interslice distance in the 38 P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45

Fig. 2. Inner morphology of the s. styloconica on the proboscis of H. virescens. Transmission electron microscopy (TEM). (A) Cross-section of a s. styloconicum at the level of the stylus showing four dendrites (D) surrounded by auxiliary cells (AC). The auxiliary cells make up two sensillar lymph cavities (SLC). (B) Cross- section of a s. styloconicum at the level of the cone showing two dendrites enveloped by the dendrite sheath (DS). (C) Longitudinal section of a cone showing three dendrites terminating approximately 250 nm proximally to the apical pore (P). (D) Longitudinal section of the same sensillum as in (C) at the level of the cone socket (Cs) showing one dendrite with a tubular body-like structure (TB) contacting the cuticle wall (CW). z-axis for the 10! objective were between 2 and 4 mm with the dry objective, the z-axis was multiplied by a factor of 1.6. The image resolution 512!512 pixels, and for the 20! and blebs at the receptor neurone terminals were reconstructed in all the 40! objectives between 1 and 2 mm with the image 15 preparations along with three chosen landmarks: the resolution 1024!1024 pixels. tritocerebral commissure and the entrance points of the two maxillary nerves. In order to obtain a three-dimensional picture of 2.5. Three-dimensional reconstructions of the brain the size and location of the whole termination area represented by the blebs, all reconstructed preparations were superimposed. A In order to determine the location of the projection area, we correct alignment was accomplished by using the three land- used the visualization software Zeiss LSM Image Examiner and marks. One brain preparation (the best) was chosen as a template, the 3D-reconstruction software Amira 3.0. Zeiss LSM Image and its surface structure was completely reconstructed. Examiner was used for visualizing the raw data of the axonal The termination area of all preparations was assembled and projections obtained in the CLSM. Amira 3.0 was used to visualised in the template brain. reconstruct the brain preparations in three dimensions and to superimpose the reconstructions, enabling an accurate compari- 3. Results son of the axonal projections between preparations. The reconstructions were made by tracing the brain structures, section 3.1. The morphology of the sensilla styloconica by section, in the anterior–posterior direction (z-axis) of the brain. To correct for the shortening in this direction caused by the Approximately 120 s. styloconica were counted on the distal refractive index mismatch in the optical pathway when using a part of the proboscis of H. virescens (Fig. 1(A)). Their P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45 39 distribution covers the area with the drinking slits where the of axons entering the SOG via the maxillary nerves. One sensilla are organized in rows along the dorsal, lateral and the pattern present in all preparations extended purely ipsilaterally. ventral side of each galea (Fig. 1(A) and (B)). Most sensillar The second pattern, observed in three of the preparations had pegs are approximately 15 mm in diameter and 60 mm long, both ipsi- and contralateral projections. becoming shorter towards the tip of the proboscis. The outer In the ipsilateral projection pattern the axons spread apart structure of this sensillum type is characterized by a proximal immediately after entering the SOG, some running dorsolat- stylus and a distal cone (Fig. 1(C)). The stylus possesses six to erally and others dorsomedially (Fig. 3(B) and (C)). The axons seven cuticular ridges extending from the narrow, cylindrical reassembled posteriorventrally to the tritocerebral commissure base to the top of the stylus surrounding the centrally located before terminating in a confined area in the dorsal SOG/ cone. The cone has a distinct socket where the cuticle is tritocerebrum, located posterior to the commisure and relatively thin (Fig. 1(D) and (E)) and a narrow tip containing ventrolaterally to the oesophagus (Fig. 3(A) and (C)). The one pore of about 200 nm in diameter (Fig. 1(F) and (G)). Out ventral branches projected medially and the dorsal branches of the 20 sensilla studied in TEM, 11 contained four dendrites laterally in the terminal area (Fig. 3(C)). In order to depict three (Fig. 2(A)) and nine contained three dendrites. Out of the five dimensionally the whole termination area associated with the sensilla studied at the cone level the dendrites of two sensilla sensory neurones of s. styloconica, the blebs at the sensory were clearly seen. Cross-sections of one cone showed two neurone terminals and the three landmarks (the two maxillary dendrites (Fig. 2(B)), and longitudinal sections of the other nerve entrances and the tritocerebral commissure) were showed three dendrites terminating approximately 250 nm reconstructed in each of the 15 preparations. The reconstruc- from the distal pore (Fig. 2(C)). At the socket level of the latter tions were subsequently superimposed and aligned according cone, one additional dendrite appeared with a distally located to the three landmarks before being placed in the tubular body-like structure contacting the cuticle wall reconstructed template brain as described in the methods (Fig. 2(D)). In the stylus of this sensillum four dendrites (Fig. 4(A)). Most of the blebs were located in the trito- were present. Each sensory neurone appeared with only one cerebral area as shown by their location posterior-ventrally dendrite process that extended through the entire sensillum to the entrance of the frontal ganglion nerve and close to the stylus enveloped by auxiliary cells (Fig. 2(A)); the outer ones tritocerebral commissure (Fig. 4(B) and (C)). A few blebs making up two sensillar lymph cavities that ran through the were also located ventrally to the commissure presumably in whole stylus and continued into the galea. These cells were the dorsal SOG (Figs. 3(C), 4(B) and (C)). However, the absent in the cone, whereas, the inner auxiliary cell continued border between the SOG and the tritocerebral neuropils is as a thick, dark sheet enveloping the dendrites up to the distal indefinable. Most blebs were located close to the anterior pore (Fig. 2(B) and (C)). surface of the neuropil. Only a few were found in a more posterior position, one as far as 120 mm posterior to the 3.2. Projections of the receptor neurones associated with tritocerebral commissure (Fig. 4(C)). sensilla styloconica In two preparations a single ipsilaterally projecting axon was stained, each of them showing a different projection To reveal the central projection pattern of the sensory pattern (Fig. 5). One axon divided in two major branches close neurones associated with the taste sensilla styloconica on the to the nerve entrance, one terminating close to the tritocerebral proboscis of H. virescens, a fluorescent dye was applied to the commissure and the midline of the brain, and the other cut end of one to five sensilla on each galea. To ensure a wide extending more laterally with terminals along a ventro-dorsal representation of sensillar location, each galea (the distal part) axis (Fig. 5(A) and (C)). According to the anterior–posterior was divided into nine regions: a proximal, medial and distal, brain axis this axon was located in the anterior part of the area each subdivided into a dorsal, lateral and ventral region defined by all blebs (Fig. 5(E)). In the second preparation the (Table 1). Out of the 36 preparations, 15 were successfully axon branched exclusively within the dorsal SOG/tritocerebral stained including 13 preparations with stained sensilla on both area with most terminals close to the midline of the brain and a galeae. In ten preparations the location of the sensilla was not few located more dorsolaterally (Fig. 5(B) and (D)). According determined. The results showed two distinct projection patterns to the anterior–posterior axis the projections of this axon were centrally in the area defined by all blebs, one branch extending anterior-dorsally and another posterior-dorsally (Fig. 5(F)). Table 1 Since each axon of this type seemed to project in a slightly Location of the stained sensilla on the distal galeae, defined as dorsally, different area, we tried to find out whether the axonal laterally and ventrally on the proximal, middle and most distal region projections in the dorsal SOG/tritocerebrum were organized Proximal Middle Distal in a topographic manner reflecting the sensillum location on Dorsal 1 (3) 1 (2) 1 (2) the proboscis (proboscotopy). The sensilla located distal- Lateral 9 (4) (3) 4 (2) laterally and proximo-laterally on the proboscis were success- Ventral (3) (2) (2) fully stained in four and nine preparations, respectively The number of preparations with successfully stained sensilla is presented. In (Table 1). A comparison was, therefore, made between the parenthesis, number of unsuccessful stained preparations. In 13 preparations locations of the blebs associated with these distal-lateral and both galeae were stained and counted as two preparations in the table. proximo-lateral sensilla (Fig. 6). On both sides the blebs 40 P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45

associated with the two categories of sensilla overlapped. The blebs on the right side showed complete overlap indicating no proboscotopy (Fig. 6(A) and (C)), whereas, the blebs on the left side showed a few non-overlapping areas indicating proboscotopy (Fig. 6(A) and (B)). In the latter case the blebs associated with the distal sensilla were located ventrally, laterally and anteriorly to the blebs associated with the proximal sensilla. The second distinct projection pattern constituted one axon with both ipsi and contralateral projections. In two of the three preparations showing this particular pattern, dye was applied to a single sensillum located proximo-laterally on the right galea. One of them is presented in Fig. 7 showing three or four axons with extensive ipsilateral projections and one branch in the contralateral SOG (Fig. 7(A)). The other preparation showed a similar bilaterally projecting neurone (not shown). Reconstruc- tion of the axon with the contralateral branch revealed that this axon entered the SOG through the right maxillary nerve and divided into one ipsi and one contralateral branch (Fig. 7(B)). Both branches arborised and terminated laterally in the SOG between the maxillary and the frontal ganglion nerve pair confined to the anterior surface of the neuropil (Fig. 7(B) and (C)). One of the ipsilateral branches extended posterior- dorsally and terminated in the centre of the area defined by the blebs in the dorsal SOG/tritocerebrum (Fig. 7(B) and (C)). In the third preparation, dye was applied to three sensilla located distal-laterally on the right galea and to three sensilla located proximo-dorsally on the left galea. This resulted in two or three stained axons in the right maxillary nerve projecting bilaterally in a similar pattern as in the two other preparations (not shown). In summary, the sensory fibres associated with s. styloconica on the proboscis were divided into two categories based on their axonal projection pattern. Category one projected exclusively ipsilaterally confined in the dorsal SOG/tritocerebrum, each axon showing small variations in branching pattern. No clear proboscotopy was observed among these neurones. Category two projected bilaterally in the SOG between the maxillary and the frontal ganglion nerve pair and was confined to the anterior surface of the neuropil. These neurones had one additional branch terminating ipsilaterally in the dorsal SOG/tritocerebrum.

Fig. 3. Central projections of sensory neurones associated with s. styloconica on the proboscis of H. virescens. Confocal laser-scanning microscopy (CLSM). (A) Projection of an optical stack showing the brain in a frontal view with optic lobes (OpL), protocerebrum (PC), oesophagus (O), tritocerebrum (T), suboesophageal ganglion (SOG) and the axons of the sensory neurones (Ax). (B) Close-up of the anterior part of the projection outlined in (A) showing the tritocerebral commissure (TC) in relation to the axons of the sensory neurones entering the SOG via the right maxillary nerve (MxN). (C) Close-up of the posterior part of the projection outlined in (A) showing the ipsilateral axonal projections in the dorsal SOG/tritocerebrum of four or ﬁve stained axons of each maxillary nerve. Note that the tritocerebral commissure is located anterior to this projection. D dorsal; V ventral. P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45 41

the tarsi and antennae elicits extension of the proboscis. The proboscis is subsequently guided into the corolla tube in the search for floral nectar. In contact with the nectar solution, the chemical composition is detected by the large number of s. styloconica on the distal part of the proboscis. Like in other lepidopteran species, the morphology of s. styloconica of H. virescens shows a large and robust hair shaft with six to seven ridges, indicating that the search for floral nectar is a rough process (Sta¨dler et al., 1974; Altner and Altner, 1986; Krenn, 1998; Walters et al., 1998). As suggested by Faucheux (1991) the stylus may serve as a tool for scrubbing the flower surface exposing the sensilla to the chemicals in the host plant tissue. Consistent with the smooth type s. styloconica in Rhodogastria bubo (Lepidoptera: Arctiidae) (Altner and Altner, 1986) approximately 50% of the s. styloconica of H. virescens contained three dendrites, whereas, four were present in the others. One of the neurones is considered to be mechanosensory, as indicated by the tubular body-like structure of the dendrite at the level of the cone socket, typical of mechanosensory dendrites innervating insect sensilla (Keil and Steinbrecht, 1984). The presence of four dendrites in the stylus and only three in the cone of the same sensillum implies that the fourth dendrite is attached to the cuticle near the cone socket. The different number of dendrites associated with the sensilla indicates that there are at least two functionally different populations of this sensillum type on the proboscis. However, functional differences may also exist between individual sensilla, as shown for the sensilla chaetica on the antenna of H. virescens, where individual variations of responses to several tastants have been found (Jørgensen pers.com). As the s. styloconica are located on the distal part, confined to the area with the drinking slits, they all provide a continuous sensory control of the food during feeding.

4.2. Projections of the receptor neurones associated with sensilla styloconica

As discussed above, gustatory and mechanosensory information is mediated separately by the two categories of sensory neurones associated with s. styloconica during inspection of a food source. Whereas, the taste neurones mediate information about food quality one might expect the mechanosensory Fig. 4. Three-dimensional reconstructions of the brain and the SOG of neurones to mediate information about food location. The H. virescens. The termination area of the sensory neurones associated with s. styloconica on the proboscis is depicted by the blebs (B), (black dots) on the axon gustatory receptor neurones might give input to interneurones terminals. (A) Frontal view of the brain with the optic lobes (OpL), antennal lobes or motor neurones involved in sucking, and the mechano- (AL) protocerebrum (PC), tritocerebrum (T), oesophagus (O) and the SOG sensory neurones to motor neurones involved in movement of neuropil (SOGn). The relative size and location of the termination area is depicted the proboscis. As shown in many insects the motor neurones by the blebs (B). (B and C) Close-up of the SOGn and the ventral part of the brain controlling the two behaviours have a different location in the showing a frontal view (B) and a lateral view (C) of the area defined by the blebs. Arrows show the direction of anterior (A) and posterior (P). MxN maxillary nerve; CNS. The motor neurones involved in the stomatogastric TC tritocerebral commissure; FN frontal ganglion nerve. system including sucking activity, seem generally to be associated with the tritocerebrum in insects (Chaudonneret, 4. Discussion 1987; Rajashekhar and Singh, 1994b; Miles and Booker, 1998). The motor neurones involved in proboscis movement 4.1. The morphology of the sensilla styloconica are generally located within or close to the SOG neuromere of the mouthpart they innervate (Altman and Kien, 1987; Rehder, When the moth Heliothis virescens encounters the flower of 1989; Rajashekhar and Singh, 1994a). According to this one a host plant, stimulation of the contact chemosensilla on might expect to find a modality specific segregation of 42 P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45

Fig. 5. The ipsilateral projection of single axons associated with s. styloconica on the proboscis of H. virescens. (A and B) Projection of two optical stacks, each shows a single axon (Ax) projecting in the dorsal SOG/tritocerebrum. (C and E) A three-dimensional reconstruction of (A) in a frontal view (C) and lateral view (E). The axon divides in two main branches close to the nerve entrance and terminates in the anterior part of the area deﬁned by the blebs. (D and F) A three-dimensional reconstruction of (B) in a frontal view (D) and lateral view (F). The axon arborises exclusively in the dorsal SOG/tritocerebrum and terminates in the central part of the area deﬁned by the blebs. The blebs (B) of ipsilateral projecting neurones from nine other preparations are superimposed in grey. T tritocerebrum;TC tritocerebral commissure; FN frontal ganglion nerve; O oesophagus; MxN maxillary nerve; SOGn SOG neuropil. P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45 43

Fig. 7. Bilateral projection of one axon associated with a s. styloconicum on the Fig. 6. Three-dimensional reconstructions of the SOG neuropil (SOGn) and the proboscis of H. virescens. (A) Projection of an optical stack showing the axonal ventral part of the brain comparing the distribution of the blebs (B) associated projections of the sensory neurones (Ax) associated with a single sensillum. with the distal s. styloconica (red) and the proximal s. styloconica (black) on the Note that one branch extends contralaterally (cb). (B) Three-dimensional proboscis of H. virescens. (A, B and C) Frontal view (A), lateral view left side reconstruction of (A) in a frontal view (B), and lateral view (C) showing the (B) and lateral view right side (C) shows segregation of the blebs associated axon (Ax) with the contralateral branch and the associated black blebs. The with the two categories of sensilla on the left side, whereas, no clear segregation grey blebs (B) on the ipsilateral side are terminals of ipsilateral projecting is observed on the right side. T tritocerebrum; TC tritocerebral commissure; neurones from seven other preparations which are merged with the projection O oesophagus; MxN maxillary nerve; FN frontal ganglion nerve. of this neurone. Note that one of the ipsilateral branches extends into the area defined by the grey blebs in the dorsal SOG/tritocerebrum. T tritocerebrum; TC the taste- and the mechanosensory projections in the CNS of tritocerebral commissure; FN frontal ganglion nerve; SOGn SOG neuropil; H. virescens, reflecting the two behaviours. Possibly the two O oesophagus; MxN maxillary nerve. different projection patterns shown in the SOG and trito- (Edgecomb and Murdock, 1992). Another possibility is that cerebrum referred to as category one and two reflect such the two different projection patterns represent specific taste segregation. The larger number of axons of category one, neurones like in Drosophila, where the proposed deterrent projecting exclusively ipsilaterally in the dorsal SOG/trito- neurones on the labellum seem to project bilaterally in the cerebrum may reflect the larger number of taste neurones of the SOG/tritocerebrum and the phagostimulant (trehalose) neur- sensilla. The axons of category two projecting bilaterally and ones project ipsilaterally (Thorne et al., 2004). In H. virescens more ventrally in the SOG might be mechanosensory. This is in one of the ipsilateral branches of the bilaterally projecting agreement with the projection pattern of the assumed axons seemed to terminate in the dorsal SOG/tritocerebrum mechanosensory neurone of the blowfly Phormia regina implying that there is a convergence of different information in 44 P. Kvello et al. / Arthropod Structure & Development 35 (2006) 35–45 this area. In comparison, a closely located area of the dorsal with the electron microscopy carried out at the Department of SOG receives projections from the proposed taste and Laboratory Medicine, Children’s and Women’s Health and at the mechanosensory neurones of the s. chaetica on the antenna Department of Biology, Norwegian University of Science and of H. virescens (Jørgensen pers.com). A convergent synaptic Technology. We acknowledge Dr Robert Brandt for his advises input of taste and mechanosensory information is shown by during the work with Amira. intracellular recordings from interneurones in the metathoracic ganglion of the locust Schistocerca gregaria and indicated in References the SOG of the fly Sarcophaga bullata (Mitchell and Itagaki, 1992; Newland, 1999). Almaas, T.J., Mustaparta, H., 1991. 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