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Anatomical organization of antennal-lobe glomeruli in males and females of the scarab beetle Holotrichia diomphalia (Coleoptera: Melolonthidae)
Arthropod Structure & Development 40 (2011) 420e428
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Anatomical organization of antennal-lobe glomeruli in males and females of the scarab beetle Holotrichia diomphalia (Coleoptera: Melolonthidae) Ji-Hua Hu, Zhi-Ying Wang, Fan Sun* School of Forestry, Northeast Forestry University, Harbin 150040, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2010 Received in revised form 13 March 2011 Accepted 16 March 2011
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. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Olfaction Glomeruli Antennal lobe Scarab beetle Dimorphism Macroglomerulus
1. Introduction The olfactory sense is essential for insects, as it plays a paramount role in locating food sources, oviposition sites, and conspecific individuals, mates included (Hartlieb and Anderson, 1999). The primary olfactory brain center of insects, the antennal lobe (AL), constitutes the first synaptic relay station of the antennal afferent pathways, as it receives input from antennal olfactory sensory neurons and sends the output to higher brain centers (Rospars, 1988; Hildebrand and Shepherd, 1997; Anton and Homberg, 1999). The building blocks of the AL found in most insect orders are the olfactory glomeruli, in which the interactions between antennal and deutocerebral neurons take place (Schachtner et al., 2005). Thus, the glomerular array is thought to constitute a chemotopic map, which ultimately leads to olfactory coding (Christensen and White, 2000; Galizia and Menzel, 2000, 2001; Ignell and Hansson, 2005). Due to their functional significance glomeruli have been anatomically mapped in several insect species. The size, number, and organization of glomeruli are shown to be conserved within individuals of one species, meaning that insects seem to have a species-specific glomerular arrangement. Their relatively small number of glomeruli, as compared to those of mammals for
* Corresponding author. E-mail address: [email protected] (F. Sun). 1467-8039/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2011.03.003
example, allows for the identification of individual glomeruli within a species (Rospars, 1988; Anton and Homberg, 1999; Schachtner et al., 2005). The numbers of glomeruli were ranging from 43 in fruit fly (Stocker, 1979, 1994; Stocker et al., 1990) to 442 in ants (workers) (Kuebler et al., 2010). In some species, as Drosophila melanogaster (Stocker, 1979, 1994; Stocker et al., 1990) and Pieris brassicae (Rospars, 1983), the glomeruli of males and females are of approximately equal size and shape. However, sexually dimorphic AL glomerular organization has also been reported in a number of Hymenoptera, Lepidoptera, and Blattaria species (Rospars, 1988; Anton and Homberg, 1999; Schachtner et al., 2005). In particular, an arrangement of enlarged glomeruli situated at the antennal nerve entrance, a so-called macroglomerulus (MG) or macroglomerular complex (MGC), has been described in males of several species. This male-specific structure is responsible for processing sex pheromone information underlying reproductive behavior (Rospars, 1983; Boeckh and Selsam, 1984; Arnold et al., 1985; Rospars and Hildebrand, 1992; Hansson, 1997). Thus far, no study has been conducted on elucidating the complete antennal-lobe glomerular organization of Coleoptera species (beetles). A few publications have reported about individually recognizable glomeruli in beetles (reviewed by Schachtner et al., 2005). Recently, Dreyer et al. (2010) described the structure and arrangement of a part of the antennal-lobe glomeruli of the red flour beetle, Tribolium castaneum. Regarding the peripheral system, the fact that semiochemicals were identified in some economically important plant-feeding scarabs (Leal, 1998) promoted studies on
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the function of olfactory receptor neurons of beetles. Thus, antennal sensilla housing receptor neurons specifically tuned to femaleproduced pheromones and plant volatiles have been identified in several scarab beetles (Renou et al., 1998; Hansson et al., 1999; Larsson et al., 2001). This information represents a foundation for investigating the structure and function of scarab beetles’ central nerve system. As severe pests during the larval stage, these insects cause substantial damage to agricultural crops and forestry productions worldwide (Lee, 2003). The significance of beetles is difficult to ignore from several points of view, the economical one not being the least. Some species of scarab beetles show an obvious sexual dimorphism of peripheral olfactory organs. This is expressed by enlarged antennal lamellae and thereby a considerably higher number of olfactory sensilla in males as compared to females (Meinecke, 1975; Ågren, 1985). For other species, the size of lamellae and number of sensilla may be similar between sexes, but the type of olfactory sensilla and/or the sensitivity of olfactory receptor cells (ORC) are significantly different (Kim and Leal, 2000; Larsson et al., 2001; Nikonov et al., 2001; Stensmyr et al., 2001). Generally, sexual dimorphism of antennal sensilla often correlates with dimorphism of the AL (Rospars, 1988). In a previous study, using scanning and transmission electron microscopes, the types and ultrastructure of antennal olfactory sensilla of Holotrichia diomphalia Bates, an important soil pest in the Northeastern region of China, was characterized. The results revealed that sensilla basciconica and sensilla placodea were two predominant types of olfactory sensilla in both sexes and that the number of sensilla was significantly higher in males than that in females (Sun et al., 2007). In this study, the anatomical organization of antennal-lobe glomeruli is described in males and females of H. diomphalia. The results, obtained by investigating serial antennal-lobe sections labeled via the reduced silver-stain technique, demonstrate a clear sexual dimorphism expressed by an enlarged male-specific macroglomerulus situated at the entennal nerve entrance. The data provide a sound foundation for functional investigations of the insect’s central nervous system. 2. Material and methods 2.1. Insects
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China), and finally mounted using Canada balsam (Shanghai molding plant specimens. Shanghai, China). Observations and photos were made by using a light microscope equipped with a 20/0.50 Ph2 dry objective lens (Olympus BX51X) and a digital camera (Olympus DP71). 2.3. Image processing and statistical analysis The stained sections of at least 20 scarab beetle were compared. Although the AL of both hemispheres were stained in some preparations, we only chose a single AL from each specimen for further analysis, disregarding possible differences between left and right hemispheres. Four brains of each sex stained with sliver-labeling technique were chosen for statistical analysis. The images were processed using Photoshop 10.0. Sections containing glomeruli were photographed in the same manner (direction and magnification) in both males and females. Adjacent sections that contain same glomeruli were matched by overlapping images from consecutive cuts, comparing shapes and locations on each image, etc. The 3-dimensional measurements of the ALs were measured (two perpendicular lengths in mm) on the section of a given glomerulus where it appears with the largest area. In the direction perpendicular to the sectioning plane, we multiplied the number of sections on which the glomerulus was visible by the section thickness (9 mm), giving a third number (depth in mm). Methods of measurements of the MG size were the same as AL. Here, the maximum length of AL and MG was measured using the DP controller 3.1.1.267 Software. All the data were analyzed with oneway ANOVA (SPSS 13.0). 2.4. Glomerular nomenclature The glomerular nomenclature used in the present study is based on the general position (Boyan et al., 1993; Nishino et al., 2005; Ghaninia et al., 2007) of single glomeruli in the AL, where two of the following capital letters denote the position; A (anterior), P (posterior), V (ventral), D (dorsal), L (lateral), M (medial), and C (central) (Figs. 1, 3e5). Positions are given according to the neuroaxis and not to the coordinates of the body. According to the sectional series from anterior to posterior, the first 9 sections of female ALs were designated as region A and the rest were
H. diomphalia eclose in the soil at the end of July and overwinter in the original place. Adults come out of overwintering in early June the following year and the adult stage lasts about 30 days. Wild adults of H. diomphalia were collected in Sun Island Park, Harbin, Heilongjiang province on June 15th, 2008, between 18:00 and 20:00. 2.2. Preparations of brain tissues for histological observation Heads of males and females were fixed in Bouin’s fixation solution (Kermel Chemical Reagent Company, Tianjin, China) for 2 days. The brains were dissected under a stereo microscope and prestained by placing them in 1% silver nitrate solution (Beijing Chemical Plant, Beijing, China) in darkness (24 h). The pre-stained brains were then embedded in paraffin and sectioned at 9 mm thickness from anterior (A) to posterior (P) or from ventral (V) to dorsal (D) with a Leica RM 2015 microtome. The serial sections were dried on histological slides before being rehydrated and stained according to Rowell’s method (Rowell, 1963). The stained sections were then dehydrated in ascending concentrations of ethanol (Restoration Technology Development Co., Ltd. Tianjin, China) (30%, 50%, 70%, 90%, 95%, 100% 3, 10 min each time), diaphanized in xylene (Kermel Chemical Reagent Company, Tianjin,
Fig. 1. Photomicrograph of the dissected brain of a female H. diomphalia Bates. AL: antennal lobe; AN: antennal nerve; CPC: circumoesophageal connective; OL: optic lobe; PT: protocerebrum; TB: tritocerebrum; A: anterior; D: dorsal; L: lateral; M: medial; P: posterior; V: ventral. Scale bar ¼ 0.5 mm.
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by their relative position to Class 1 glomeruli. Class 3 glomeruli are poorly demarcated ones with variation in more than one criterion.
3. Results 3.1. The antennal lobe The ALs of H. diomphalia are located at the anterior part of the deutocerebrum in a left and right symmetric manner. The chemosensory axons which constitute the antennal nerve, project to the ipsilateral AL (Fig. 1). The stainings show, in both sexes, cell bodies distributed at the periphery of the AL and an array of glomeruli arranged in one to two layers around a central core (Fig. 4EeG and Fig. 5FeG). Individual glomeruli are separated by glia and form irregular spherical units (Figs. 4 and 5).
Fig. 2. One section of a H. diomphalia Bates brain. AL: antennal lobe; CB: central body; MI: medulla interna; PB: protocerebral bridge.
designated as region P as glomeruli were present in 18 consecutive sectional planes of female ALs. Similarly, the first 12 sections of male ALs were designated as region A and the rest of 12 sections were designated as region P as glomeruli were present in 24 consecutive sectional planes of male ALs. Two circles were drawn with a common center located at the middle point of the maximum diameter of the AL (the large circle diameter was the maximum diameter of AL and the small circle diameter was half of the large circle diameter). The small circle represented C region. The area between the large and small circle was divided into 4 equal parts and designed as sub-regions D, L, V, and M (Fig. 3). The number in each glomerular name represents the sequence when it appeared in that specific region, e.g. AD8 means that this glomerulus appeared in the AD region in the 8th sectional plane. Glomeruli with lower number in region A are located more anteriorly in the antennal lobe whereas high numbers in the P region indicates glomeruli positioned posteriorly (Fig. 2). Glomeruli were categorized into 3 classes as described by Ghaninia et al. (2007). Briefly, class 1 glomeruli are fairly constant in shape, size, position, brightness, and distinctiveness. Class 2 glomeruli generally comply with Class 1 criteria and are identified
Fig. 3. The naming of glomeruli in different regions. The larger/outside circle represents the maximum diameter of the antennal lobe. The smaller/inside circle, drawn at a diameter that is half of the larger circle diameter, defines the central region (region C). The area between the large and small circle is divided into 4 equal parts and designed as sub-regions D, L, V, and M.
3.2. Sexual dimorphism There is a pronounced sexual dimorphism concerning glomerular arrangement. The males have, in addition to an assembly of ordinary glomeruli, one cap-shaped MG occupying the entire AM region and accounting for about 1/3 of the AL(Fig. 5BeG). Females, on the other hand, have an assembly of similarly sized glomeruli only(Fig. 4). In the females, these ordinary units are arranged around a central core devoid of glomeruli whereas the corresponding glomeruli of the males partly occupy this region due to the presence of an enlarged MG. Additionally, the glomeruli at the posterior part of male ALs were more densely packed and individual glomeruli were difficult to delineate when compared with females. Furthermore, male ALs were relatively larger than those of females as indicated by the significant difference (P ¼ 0.004) in length at the AeP direction. The lengths along directions of MeL and DeV, respectively, were not significantly different (P ¼ 0.13 and 0.11 respectively; Table 1).
3.3. Description and discrimination of antennal glomeruli From analyzing the silver-stained sections of each individual, a total number of approximately 60 antennal-lobe glomeruli were estimated in females of H. diomphalia. Thus, the 4 stained section series of ALs comprised 59, 59, 61, and 62 glomeruli, respectively. The glomeruli in the specimen containing 61 units, units with names given according to the standard previously described, resulted in 36 Class 1 glomeruli, 18 Class 2 glomeruli, and 8 Class 3 glomeruli (Table 2, Fig. 4). Glomeruli of Class 3 are divided into 4 groups, AD3 and AD5(Fig. 4C), AD6 and AC6(Fig. 4C), PD2 and PD3(Fig. 4H),PC5 and PC6(Fig. 4I). The two glomeruli of each group were arranged closely to each other and their boundaries were blurred. Regarding the male preparations, the cap-shaped MG were observed in all of the 4 stained section serials respectively. The 3-D dimensions of the MG at their maximum were 131 4.68 mm (AeV), 81.6 3.61 mm (LeM),126 5.87 (AeP) (Mean MS,n ¼ 4 specimens). But the glomeruli at the posterior part of male ALs were more densely packed and individual glomeruli were difficult to delineate because of their unclear boundary(Fig. 5HeJ). The glomeruli at the anteriorly part units, were distinguishable and given names according to the standard previously described, resulted in 23 Class 1 glomeruli, 14 Class 2 glomeruli (Table 2, Fig. 5BeG).
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Fig. 4. Anterior view of the antennal lobe of a female H. diomphalia Bates, stained by the silver-labeling technique. To display all glomeruli in a limited number of images, 10 sections at different intervals, from anterior to posterior, are selected from a total of 18 images. The depth within the AL neuropil is indicated in the upper-right corner of each image. Anterior is up and lateral to the right. The left column displays unaltered silver-stained preparations (raw data). The middle column displays demarcated glomeruli superimposed on gray scale inverted images. The right column displays identified glomeruli. Scale bar ¼ 100 mm.
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Fig. 4. (continued).
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Fig. 5. Anterior view of the part of antennal lobe of a male H. diomphalia Bates, stained by the silver-labeling technique. To display all glomeruli in a limited number of images, 10 sections from anterior to posterior were selected from a total of 24 images at different intervals, where the depth within the AL neuropil is indicated in the upper-right corner of each image, in order to display all glomeruli and to restrict the number of sections displayed. Anterior is up and lateral to the right. From A to G, left column displays unaltered silver-stain (raw data). Middle column displays demarcated glomeruli superimposed on gray scale inverted images. Right column displays identified glomeruli. H, I and J displays unaltered silver-stain (raw data) near the posterior end. Scale bar ¼ 100 mm. For colour, please refer to the online version of this article.
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Fig. 5. (continued).
4. Discussion 4.1. Sexual dimorphism Sexual dimorphic features in the AL (number, location, and arrangement of dimorphic glomeruli) differ considerably in various insect orders, suggesting that parallel convergent evolution has occurred toward sexual dimorphic structures in these taxa as a consequence of the involvement of the olfactory system in sexual communication and mate finding (Schachtner et al., 2005). The presence of male-specific enlarged glomeruli (macroglomeruli) is the most common form of sexual dimorphism, occurring in cockroaches, honey bees, ants, and moths. The appearance of male-specific enlarged glomeruli has been observed mainly in species with dimorphic antennae and whose males are attracted to females by olfactory signals (Rospars, 1988). One of the most thoroughly studied species, the male tobacco hornworm, Manduca sexta, possesses a special MGC that correlates with the presence of long, type-I trichoid sensilla that are found exclusively on male antennae. These sensilla house two types of ORCs, each of which is sensitive to one of the two key components of the conspecific female moth’s sex pheromone (Kaissling et al., 1989; Keil, 1989; Lee and Strausfeld, 1990). Similar to moths, many female
scarab beetle species release sex pheromones to attract mates at long distances (Leal, 1998) and sex-specific characteristics of antennal sensilla are quite common (Sun et al., 2007). The males of H. diomphalia have a considerably larger number(9)of sensilla basiconca on their antenna as compared to females (Sun et al., 2007). Therefore, it is plausible to hypothesize that these sensilla serve as an organ for detection of female-produced sex pheromones. Regarding H. diomphalia, the observation that females extend a white colored spherical saccule at the abdominal tip, suggests a sexual behavior including release of chemical signals to attract males for mating. Thus, we speculate that the male-specific MG identified in the current study receives input from pheromone-detecting receptor neurons housed by the sensilla basiconica. Table 1 Size comparisons of female and male antennal lobe of Holotrichia diomphalia. Direction
Female(mm)
Male(mm)
MeL DeV AeP
163.00 10.96 A 181.40 9.28 A 160.20 10.67 A
196.00 9.25 A 228.50 21.59 A 226.75 11.49 B
Data are presented as Mean MS (n ¼ 4 specimens). Means within a row followed by the same letter were not significantly different at the level of 0.05.
J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428 Table 2 Glomerular neuropils in the antennal lobe of female and male Holotrichia diomphalia by the silver-labeling technique. Malea
Female Glomerulus
Classb
AC1 AC2 AC3 AC4 AC5 AC6 AC7 AC8 AD1 AD2 AD3 AD4 AD5 AD6 AD7 AD8 AD9 AD10 AD11 AL1 AL2 AL3 AL4 AL5 AL6 AL7 AL8 AM1 AM2 AM3 AM4 AM5 AM6 AV1 AV2 AV3 AV4 AV5 AV6 PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PD1 PD2 PD3 PL1 PL2 PM1 PM2 PM3 PV1 PV2 PV3 PV4
1 2 2 1 1 3 1 2 1 1 3 1 3 3 2 1 2 2 2 1 1 1 1 1 1 2 2 1 1 1 2 2 1 1 1 1 1 1 2 2 1 1 1 3 3 1 1 1 1 1 3 3 1 1 2 1 2 1 2 2 1
Commentc d d
sh, p sz
sh, sz, p sz, p sh, p sh sh sh, d d
d d
d d
Glomerulus
Classb
AC1 AC2 AC3 AC4 AC5 AC6 AC7 AC8 AC9 AC10 AC11 AC12 AC13 AD1 AD2 AD3 AD4 AL1 AL2 AL3 AL4 AL5 AL6 AL7 AL8 AL9 AV1 AV2 AV3 AV4 AV5 AV6 AV7 AV8 PD1 PD2 MG
1 1 1 1 1 2 1 1 1 1 2 2 1 1 1 1 2 1 1 1 2 2 1 2 1 2 1 1 1 2 2 2 2 2 1 2 1
Commentc
sh
sh sh
sz
d d sh d
sh, d sz, d sz, d sh, d sh, d sh
sh sh
sh, sz, p sh, sz, p
sh, sz, p sh, sz, p
sh, d sh, d sh sh
a
Comprising only the glomeruli located at the anterior antennal lobe of male. Classes refer to the distinctness of glomeruli (for details see Section 2). c Deviation(s) from class distinctness is indicated under comment: sh: shape; sz: size; p: position; d: demarcation. b
In male moths the MGC consists of 2e4 glomeruli positioned close to the entrance of the antennal nerve. Each of these malespecific units receive input from a population of ORNs that detect one particular constituent of female-produced semiochemicals (Hansson et al., 1992; Ochieng et al., 1995; Todd et al., 1995; Berg
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et al., 1998, 2005; Lee et al., 2006a). Thus, the number of macroglomerular sub-structures seems to be correlated with the number of physiological male-specific receptor neuron categories. However, there also are exceptions, e.g. in Helicoverpa zea, ORNs that detect different semiochemicals are reported to project to a single MGC subunit (Lee et al., 2006b). Moreover, in Lobesia botrana and Cydia pomonella (Tortricidea), only one large glomerulus is present in the ALs of males, while three different components serve as pheromones (Masante-Roca et al., 2005; Varela et al., 2009). Also in the cockroach, Periplaneta americana, two types of pheromone receptor neurons tuned to the female-produced attractants periplanone A and periplanone B, respectively, are reported to target one malespecific unit, the MG (Boeckh et al., 1970; Boeckh and Selsam, 1984). The results presented here, demonstrate the presence of a male-specific antennal-lobe structure of H. diomphalia, an MG similar to that of cockroaches. The prominence of the MG in this Coleopteran species, both as concern size and position, indicates the importance of pheromone signals for reproductive behavior. 4.2. Antennal glumeruli of H. diomphalia: number and description The number of approximately 60 antennal-lobe glomeruli in H. diomphalia, as determined for the females in the present study, is in correspondence with previous reports from other insect species. In particular, this number is similar to the finding of ca 70 glomeruli in the red flour beetle T. castaneum (Dreyer et al., 2010). As compared to mammals, which often have thousands of glomeruli in their primary olfactory center (Menco, 1980), insects usually possess a considerably smaller number of typically 50e250 glomeruli (Rospars, 1988; Anton and Homberg, 1999; Schachtner et al., 2005). It seems that the numbers of glomeruli are similar across species of the same insect group. Thus, Diptera generally have numbers ranging from 40 to 50, Lepidoptera 60 to 65, and Hymenoptera more than 100 (Schachtner et al., 2005; Ignell and Hansson, 2005; Ghaninia et al., 2007; Masante-Roca et al., 2005; Varela et al., 2009; Nishikawa et al., 2008; Zube and Rössler, 2008). Although we numbered and named all found glomeruli, one must recognize that it is an estimation. Two factors could affect this estimation. One is that smaller glomeruli may not be visible due to the thickness of the sections causing a smaller estimation. The other is that the unclear boundary of Class 3 glomeruli may increase the estimation. Total of 8 Class 3 glomeruli were identified in females. Unclear boundaries were present between AD3 and AD5, AD6 and AC6, PC5 and PC6, PD2 and PD3. Therefore, it is possible that the pairs we named in the study are actual one glomerulus. These two factors are the likely reasons for the discrepancy of number of glomeruli in the four samples. In males the glomeruli at the posterior part of male ALs were more densely packed and individual glomeruli were difficult to delineate because of the unclear boundary. This matter of fact may of course be due to technical problems concerning penetration of dye in the deeper brain layers. However, the distinctiveness of glomeruli may also be closely related to the composition of the neural network, i.e., the density of neuropil. The number of afferent neurons in posteriorly located glomeruli may for instance be lower than that of more anteriorly positioned glomeruli (Ghaninia et al., 2007). Regardless of the cause, the estimates should be improved by alternative staining methods or novel molecular techniques in order to verify and complement the presented data. Acknowledgments We acknowledge Dr. Bente G. Berg (Norwegian University of Science and Technology) for critically reading and greatly improving the manuscript. We also thank two anonymous reviewers for their
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Three-dimensional antennal lobe atlas of male and female moths, Lobesia botrana (Lepidoptera: Tortricidae) and glomerular representation of plant volatiles in females. Journal of Experimental Biology 208, 1147e1159. Meinecke, C.C., 1975. Riechsensilla und Systematik der Lamellicornia (Insecta, Coleoptera). Zoomorphologie 82, 1e42. Menco, BPhM, 1980. Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog, ox, rat, and dog.III. Tightjunctions. Cell Tissue Research 211, 361e373. Nikonov, A., Valiyaveettil, J.T., Leal, W.S., 2001. A photoaffinity-labeled green leaf volatile compound ‘tricks’ highly selective and sensitive insect olfactory receptor neurons. Chemical Senses 26, 49e54. Nishikawa, M., Nishino, H., Misaka, Y., Kubota, M., Tsuji, E., Satoji, Y., Ozaki, M., Yokohari, F., 2008. Sexual dimorphism in the antennal lobe of the ant Camponotus japonicus. Zoological Science 25, 195e204. Nishino, H., Nishikawa, M., Yokohari, F., Mizunami, M., 2005. Dual, multilayered somatosensory maps formed by tactile and contact chemosensory afferents in an insect brain. Journal of Comparative Neurology 493, 291e308. Ochieng, S.A., Anderson, P., Hansson, B.S., 1995. Antennal lobe projection patterns of olfactory receptor neurons involved in sex pheromone detection in Spodoptera littoralis (Lepidoptera: Noctuidae). Tissue Cell 27, 221e232. Renou, M., Tauban, D., Morin, J.P., 1998. Structure and function of antennal pore plate sensilla of Oryctes rhinoceros (L.) (Coleoptera: Dynastidae). International Journal of Insect Morphology & Embryology 27, 227e233. Rospars, J.P., 1983. Invariance and sex-specific variations of the glomerular organization in the antennal lobes of a moth, Mamestra brassicae and a butterfly, Pieris brassicae. Journal of Comparative Neurology 220, 80e96. Rospars, J.P., 1988. Structure and development of the insect antennodeuto-cerebral system. International Journal of Insect Morphology & Embryology 17, 243e294. Rospars, J.P., Hildebrand, J.G., 1992. Anatomical identification of glomeruli in the antennal lobes of the male sphinx moth Manduca sexta. Cell Tissue Research 270, 205e227. Rowell, C.H.F., 1963. A general method for silvering invertebrate nervous systems. Quarterly Journal of Microsopical Science 104 (65), 81e87. Schachtner, J., Schmidt, M., Homberg, U., 2005. Organization and evolutionary trends of primary olfactory brain centers in Tetraconata (Crustacea þ Hexapoda). Arthropod Structure & Development 34, 257e299. Stensmyr, M.C., Larsson, M.C., Bice, S., Hansson, B.S., 2001. Detection of fruit and flower emitted volatiles by olfactory receptor neurons in the polyphagous fruit chafer Pachnoda marginata (Coleoptera: Cetoniinae). Journal of Comparative & Physiological Psychology A 187, 509e519. Stocker, R.F., 1979. Fine structural comparison of the antennal nerve in the homeotic mutant antennapedia with the wild-type antennal and second leg nerves of Drosophila melanogaster. Journal of Morphology 160, 209e222. Stocker, R.F., 1994. The organization of the chemosensory system in Drosophila melanogaster: a review. Cell Tissue Research 275, 3e26. Stocker, R.F., Lienhard, M.C., Borst, A., Fischbach, K.F., 1990. Neuronal architecture of the antennal lobe in Drosophila melanogaster. Cell Tissue Research 262, 9e34. Sun, F., Hu, J.-H., Wang, G.-L., Peng, L., 2007. Ultrastructure of olfactory sensilla of Holotrichia diomphalia bates (Coleoptera: Melolonthidae). Acta Entomologica Sinica 50, 675e681 (in Chinese). Todd, J.L., Anton, S., Hansson, B.S., Baker, T.C., 1995. Functional organization of the macroglomerular complex related to behaviorally expressed olfactory redundancy in male cabbage looper moth. Physiological Enomology 20, 349e361. Varela, N., Couton, L., Gemeno, C., Avilla, J., Rospars, J.P., Anton, S., 2009. Threedimensional antennal lobe atlas of the oriental fruit moth, Cydia molesta (Busck) (Lepidoptera: Tortricidae): comparison of male and female glomerular organization. Cell and Tissue Research 337, 513e526. Zube, C., Rössler, W., 2008. Caste and sex specific adaptations within the olfactory pathway in the brain of the ant Camponotus floridanus. Arthropod Structure & Development 37, 469e479.
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Anatomical organization of antennal-lobe glomeruli in males and females of the scarab beetle Holotrichia diomphalia (Coleoptera: Melolonthidae) Ji-Hua Hu, Zhi-Ying Wang, Fan Sun* School of Forestry, Northeast Forestry University, Harbin 150040, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2010 Received in revised form 13 March 2011 Accepted 16 March 2011
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. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Olfaction Glomeruli Antennal lobe Scarab beetle Dimorphism Macroglomerulus
1. Introduction The olfactory sense is essential for insects, as it plays a paramount role in locating food sources, oviposition sites, and conspecific individuals, mates included (Hartlieb and Anderson, 1999). The primary olfactory brain center of insects, the antennal lobe (AL), constitutes the first synaptic relay station of the antennal afferent pathways, as it receives input from antennal olfactory sensory neurons and sends the output to higher brain centers (Rospars, 1988; Hildebrand and Shepherd, 1997; Anton and Homberg, 1999). The building blocks of the AL found in most insect orders are the olfactory glomeruli, in which the interactions between antennal and deutocerebral neurons take place (Schachtner et al., 2005). Thus, the glomerular array is thought to constitute a chemotopic map, which ultimately leads to olfactory coding (Christensen and White, 2000; Galizia and Menzel, 2000, 2001; Ignell and Hansson, 2005). Due to their functional significance glomeruli have been anatomically mapped in several insect species. The size, number, and organization of glomeruli are shown to be conserved within individuals of one species, meaning that insects seem to have a species-specific glomerular arrangement. Their relatively small number of glomeruli, as compared to those of mammals for
* Corresponding author. E-mail address: [email protected] (F. Sun). 1467-8039/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2011.03.003
example, allows for the identification of individual glomeruli within a species (Rospars, 1988; Anton and Homberg, 1999; Schachtner et al., 2005). The numbers of glomeruli were ranging from 43 in fruit fly (Stocker, 1979, 1994; Stocker et al., 1990) to 442 in ants (workers) (Kuebler et al., 2010). In some species, as Drosophila melanogaster (Stocker, 1979, 1994; Stocker et al., 1990) and Pieris brassicae (Rospars, 1983), the glomeruli of males and females are of approximately equal size and shape. However, sexually dimorphic AL glomerular organization has also been reported in a number of Hymenoptera, Lepidoptera, and Blattaria species (Rospars, 1988; Anton and Homberg, 1999; Schachtner et al., 2005). In particular, an arrangement of enlarged glomeruli situated at the antennal nerve entrance, a so-called macroglomerulus (MG) or macroglomerular complex (MGC), has been described in males of several species. This male-specific structure is responsible for processing sex pheromone information underlying reproductive behavior (Rospars, 1983; Boeckh and Selsam, 1984; Arnold et al., 1985; Rospars and Hildebrand, 1992; Hansson, 1997). Thus far, no study has been conducted on elucidating the complete antennal-lobe glomerular organization of Coleoptera species (beetles). A few publications have reported about individually recognizable glomeruli in beetles (reviewed by Schachtner et al., 2005). Recently, Dreyer et al. (2010) described the structure and arrangement of a part of the antennal-lobe glomeruli of the red flour beetle, Tribolium castaneum. Regarding the peripheral system, the fact that semiochemicals were identified in some economically important plant-feeding scarabs (Leal, 1998) promoted studies on
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the function of olfactory receptor neurons of beetles. Thus, antennal sensilla housing receptor neurons specifically tuned to femaleproduced pheromones and plant volatiles have been identified in several scarab beetles (Renou et al., 1998; Hansson et al., 1999; Larsson et al., 2001). This information represents a foundation for investigating the structure and function of scarab beetles’ central nerve system. As severe pests during the larval stage, these insects cause substantial damage to agricultural crops and forestry productions worldwide (Lee, 2003). The significance of beetles is difficult to ignore from several points of view, the economical one not being the least. Some species of scarab beetles show an obvious sexual dimorphism of peripheral olfactory organs. This is expressed by enlarged antennal lamellae and thereby a considerably higher number of olfactory sensilla in males as compared to females (Meinecke, 1975; Ågren, 1985). For other species, the size of lamellae and number of sensilla may be similar between sexes, but the type of olfactory sensilla and/or the sensitivity of olfactory receptor cells (ORC) are significantly different (Kim and Leal, 2000; Larsson et al., 2001; Nikonov et al., 2001; Stensmyr et al., 2001). Generally, sexual dimorphism of antennal sensilla often correlates with dimorphism of the AL (Rospars, 1988). In a previous study, using scanning and transmission electron microscopes, the types and ultrastructure of antennal olfactory sensilla of Holotrichia diomphalia Bates, an important soil pest in the Northeastern region of China, was characterized. The results revealed that sensilla basciconica and sensilla placodea were two predominant types of olfactory sensilla in both sexes and that the number of sensilla was significantly higher in males than that in females (Sun et al., 2007). In this study, the anatomical organization of antennal-lobe glomeruli is described in males and females of H. diomphalia. The results, obtained by investigating serial antennal-lobe sections labeled via the reduced silver-stain technique, demonstrate a clear sexual dimorphism expressed by an enlarged male-specific macroglomerulus situated at the entennal nerve entrance. The data provide a sound foundation for functional investigations of the insect’s central nervous system. 2. Material and methods 2.1. Insects
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China), and finally mounted using Canada balsam (Shanghai molding plant specimens. Shanghai, China). Observations and photos were made by using a light microscope equipped with a 20/0.50 Ph2 dry objective lens (Olympus BX51X) and a digital camera (Olympus DP71). 2.3. Image processing and statistical analysis The stained sections of at least 20 scarab beetle were compared. Although the AL of both hemispheres were stained in some preparations, we only chose a single AL from each specimen for further analysis, disregarding possible differences between left and right hemispheres. Four brains of each sex stained with sliver-labeling technique were chosen for statistical analysis. The images were processed using Photoshop 10.0. Sections containing glomeruli were photographed in the same manner (direction and magnification) in both males and females. Adjacent sections that contain same glomeruli were matched by overlapping images from consecutive cuts, comparing shapes and locations on each image, etc. The 3-dimensional measurements of the ALs were measured (two perpendicular lengths in mm) on the section of a given glomerulus where it appears with the largest area. In the direction perpendicular to the sectioning plane, we multiplied the number of sections on which the glomerulus was visible by the section thickness (9 mm), giving a third number (depth in mm). Methods of measurements of the MG size were the same as AL. Here, the maximum length of AL and MG was measured using the DP controller 3.1.1.267 Software. All the data were analyzed with oneway ANOVA (SPSS 13.0). 2.4. Glomerular nomenclature The glomerular nomenclature used in the present study is based on the general position (Boyan et al., 1993; Nishino et al., 2005; Ghaninia et al., 2007) of single glomeruli in the AL, where two of the following capital letters denote the position; A (anterior), P (posterior), V (ventral), D (dorsal), L (lateral), M (medial), and C (central) (Figs. 1, 3e5). Positions are given according to the neuroaxis and not to the coordinates of the body. According to the sectional series from anterior to posterior, the first 9 sections of female ALs were designated as region A and the rest were
H. diomphalia eclose in the soil at the end of July and overwinter in the original place. Adults come out of overwintering in early June the following year and the adult stage lasts about 30 days. Wild adults of H. diomphalia were collected in Sun Island Park, Harbin, Heilongjiang province on June 15th, 2008, between 18:00 and 20:00. 2.2. Preparations of brain tissues for histological observation Heads of males and females were fixed in Bouin’s fixation solution (Kermel Chemical Reagent Company, Tianjin, China) for 2 days. The brains were dissected under a stereo microscope and prestained by placing them in 1% silver nitrate solution (Beijing Chemical Plant, Beijing, China) in darkness (24 h). The pre-stained brains were then embedded in paraffin and sectioned at 9 mm thickness from anterior (A) to posterior (P) or from ventral (V) to dorsal (D) with a Leica RM 2015 microtome. The serial sections were dried on histological slides before being rehydrated and stained according to Rowell’s method (Rowell, 1963). The stained sections were then dehydrated in ascending concentrations of ethanol (Restoration Technology Development Co., Ltd. Tianjin, China) (30%, 50%, 70%, 90%, 95%, 100% 3, 10 min each time), diaphanized in xylene (Kermel Chemical Reagent Company, Tianjin,
Fig. 1. Photomicrograph of the dissected brain of a female H. diomphalia Bates. AL: antennal lobe; AN: antennal nerve; CPC: circumoesophageal connective; OL: optic lobe; PT: protocerebrum; TB: tritocerebrum; A: anterior; D: dorsal; L: lateral; M: medial; P: posterior; V: ventral. Scale bar ¼ 0.5 mm.
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by their relative position to Class 1 glomeruli. Class 3 glomeruli are poorly demarcated ones with variation in more than one criterion.
3. Results 3.1. The antennal lobe The ALs of H. diomphalia are located at the anterior part of the deutocerebrum in a left and right symmetric manner. The chemosensory axons which constitute the antennal nerve, project to the ipsilateral AL (Fig. 1). The stainings show, in both sexes, cell bodies distributed at the periphery of the AL and an array of glomeruli arranged in one to two layers around a central core (Fig. 4EeG and Fig. 5FeG). Individual glomeruli are separated by glia and form irregular spherical units (Figs. 4 and 5).
Fig. 2. One section of a H. diomphalia Bates brain. AL: antennal lobe; CB: central body; MI: medulla interna; PB: protocerebral bridge.
designated as region P as glomeruli were present in 18 consecutive sectional planes of female ALs. Similarly, the first 12 sections of male ALs were designated as region A and the rest of 12 sections were designated as region P as glomeruli were present in 24 consecutive sectional planes of male ALs. Two circles were drawn with a common center located at the middle point of the maximum diameter of the AL (the large circle diameter was the maximum diameter of AL and the small circle diameter was half of the large circle diameter). The small circle represented C region. The area between the large and small circle was divided into 4 equal parts and designed as sub-regions D, L, V, and M (Fig. 3). The number in each glomerular name represents the sequence when it appeared in that specific region, e.g. AD8 means that this glomerulus appeared in the AD region in the 8th sectional plane. Glomeruli with lower number in region A are located more anteriorly in the antennal lobe whereas high numbers in the P region indicates glomeruli positioned posteriorly (Fig. 2). Glomeruli were categorized into 3 classes as described by Ghaninia et al. (2007). Briefly, class 1 glomeruli are fairly constant in shape, size, position, brightness, and distinctiveness. Class 2 glomeruli generally comply with Class 1 criteria and are identified
Fig. 3. The naming of glomeruli in different regions. The larger/outside circle represents the maximum diameter of the antennal lobe. The smaller/inside circle, drawn at a diameter that is half of the larger circle diameter, defines the central region (region C). The area between the large and small circle is divided into 4 equal parts and designed as sub-regions D, L, V, and M.
3.2. Sexual dimorphism There is a pronounced sexual dimorphism concerning glomerular arrangement. The males have, in addition to an assembly of ordinary glomeruli, one cap-shaped MG occupying the entire AM region and accounting for about 1/3 of the AL(Fig. 5BeG). Females, on the other hand, have an assembly of similarly sized glomeruli only(Fig. 4). In the females, these ordinary units are arranged around a central core devoid of glomeruli whereas the corresponding glomeruli of the males partly occupy this region due to the presence of an enlarged MG. Additionally, the glomeruli at the posterior part of male ALs were more densely packed and individual glomeruli were difficult to delineate when compared with females. Furthermore, male ALs were relatively larger than those of females as indicated by the significant difference (P ¼ 0.004) in length at the AeP direction. The lengths along directions of MeL and DeV, respectively, were not significantly different (P ¼ 0.13 and 0.11 respectively; Table 1).
3.3. Description and discrimination of antennal glomeruli From analyzing the silver-stained sections of each individual, a total number of approximately 60 antennal-lobe glomeruli were estimated in females of H. diomphalia. Thus, the 4 stained section series of ALs comprised 59, 59, 61, and 62 glomeruli, respectively. The glomeruli in the specimen containing 61 units, units with names given according to the standard previously described, resulted in 36 Class 1 glomeruli, 18 Class 2 glomeruli, and 8 Class 3 glomeruli (Table 2, Fig. 4). Glomeruli of Class 3 are divided into 4 groups, AD3 and AD5(Fig. 4C), AD6 and AC6(Fig. 4C), PD2 and PD3(Fig. 4H),PC5 and PC6(Fig. 4I). The two glomeruli of each group were arranged closely to each other and their boundaries were blurred. Regarding the male preparations, the cap-shaped MG were observed in all of the 4 stained section serials respectively. The 3-D dimensions of the MG at their maximum were 131 4.68 mm (AeV), 81.6 3.61 mm (LeM),126 5.87 (AeP) (Mean MS,n ¼ 4 specimens). But the glomeruli at the posterior part of male ALs were more densely packed and individual glomeruli were difficult to delineate because of their unclear boundary(Fig. 5HeJ). The glomeruli at the anteriorly part units, were distinguishable and given names according to the standard previously described, resulted in 23 Class 1 glomeruli, 14 Class 2 glomeruli (Table 2, Fig. 5BeG).
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Fig. 4. Anterior view of the antennal lobe of a female H. diomphalia Bates, stained by the silver-labeling technique. To display all glomeruli in a limited number of images, 10 sections at different intervals, from anterior to posterior, are selected from a total of 18 images. The depth within the AL neuropil is indicated in the upper-right corner of each image. Anterior is up and lateral to the right. The left column displays unaltered silver-stained preparations (raw data). The middle column displays demarcated glomeruli superimposed on gray scale inverted images. The right column displays identified glomeruli. Scale bar ¼ 100 mm.
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Fig. 4. (continued).
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425
Fig. 5. Anterior view of the part of antennal lobe of a male H. diomphalia Bates, stained by the silver-labeling technique. To display all glomeruli in a limited number of images, 10 sections from anterior to posterior were selected from a total of 24 images at different intervals, where the depth within the AL neuropil is indicated in the upper-right corner of each image, in order to display all glomeruli and to restrict the number of sections displayed. Anterior is up and lateral to the right. From A to G, left column displays unaltered silver-stain (raw data). Middle column displays demarcated glomeruli superimposed on gray scale inverted images. Right column displays identified glomeruli. H, I and J displays unaltered silver-stain (raw data) near the posterior end. Scale bar ¼ 100 mm. For colour, please refer to the online version of this article.
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Fig. 5. (continued).
4. Discussion 4.1. Sexual dimorphism Sexual dimorphic features in the AL (number, location, and arrangement of dimorphic glomeruli) differ considerably in various insect orders, suggesting that parallel convergent evolution has occurred toward sexual dimorphic structures in these taxa as a consequence of the involvement of the olfactory system in sexual communication and mate finding (Schachtner et al., 2005). The presence of male-specific enlarged glomeruli (macroglomeruli) is the most common form of sexual dimorphism, occurring in cockroaches, honey bees, ants, and moths. The appearance of male-specific enlarged glomeruli has been observed mainly in species with dimorphic antennae and whose males are attracted to females by olfactory signals (Rospars, 1988). One of the most thoroughly studied species, the male tobacco hornworm, Manduca sexta, possesses a special MGC that correlates with the presence of long, type-I trichoid sensilla that are found exclusively on male antennae. These sensilla house two types of ORCs, each of which is sensitive to one of the two key components of the conspecific female moth’s sex pheromone (Kaissling et al., 1989; Keil, 1989; Lee and Strausfeld, 1990). Similar to moths, many female
scarab beetle species release sex pheromones to attract mates at long distances (Leal, 1998) and sex-specific characteristics of antennal sensilla are quite common (Sun et al., 2007). The males of H. diomphalia have a considerably larger number(9)of sensilla basiconca on their antenna as compared to females (Sun et al., 2007). Therefore, it is plausible to hypothesize that these sensilla serve as an organ for detection of female-produced sex pheromones. Regarding H. diomphalia, the observation that females extend a white colored spherical saccule at the abdominal tip, suggests a sexual behavior including release of chemical signals to attract males for mating. Thus, we speculate that the male-specific MG identified in the current study receives input from pheromone-detecting receptor neurons housed by the sensilla basiconica. Table 1 Size comparisons of female and male antennal lobe of Holotrichia diomphalia. Direction
Female(mm)
Male(mm)
MeL DeV AeP
163.00 10.96 A 181.40 9.28 A 160.20 10.67 A
196.00 9.25 A 228.50 21.59 A 226.75 11.49 B
Data are presented as Mean MS (n ¼ 4 specimens). Means within a row followed by the same letter were not significantly different at the level of 0.05.
J.-H. Hu et al. / Arthropod Structure & Development 40 (2011) 420e428 Table 2 Glomerular neuropils in the antennal lobe of female and male Holotrichia diomphalia by the silver-labeling technique. Malea
Female Glomerulus
Classb
AC1 AC2 AC3 AC4 AC5 AC6 AC7 AC8 AD1 AD2 AD3 AD4 AD5 AD6 AD7 AD8 AD9 AD10 AD11 AL1 AL2 AL3 AL4 AL5 AL6 AL7 AL8 AM1 AM2 AM3 AM4 AM5 AM6 AV1 AV2 AV3 AV4 AV5 AV6 PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PD1 PD2 PD3 PL1 PL2 PM1 PM2 PM3 PV1 PV2 PV3 PV4
1 2 2 1 1 3 1 2 1 1 3 1 3 3 2 1 2 2 2 1 1 1 1 1 1 2 2 1 1 1 2 2 1 1 1 1 1 1 2 2 1 1 1 3 3 1 1 1 1 1 3 3 1 1 2 1 2 1 2 2 1
Commentc d d
sh, p sz
sh, sz, p sz, p sh, p sh sh sh, d d
d d
d d
Glomerulus
Classb
AC1 AC2 AC3 AC4 AC5 AC6 AC7 AC8 AC9 AC10 AC11 AC12 AC13 AD1 AD2 AD3 AD4 AL1 AL2 AL3 AL4 AL5 AL6 AL7 AL8 AL9 AV1 AV2 AV3 AV4 AV5 AV6 AV7 AV8 PD1 PD2 MG
1 1 1 1 1 2 1 1 1 1 2 2 1 1 1 1 2 1 1 1 2 2 1 2 1 2 1 1 1 2 2 2 2 2 1 2 1
Commentc
sh
sh sh
sz
d d sh d
sh, d sz, d sz, d sh, d sh, d sh
sh sh
sh, sz, p sh, sz, p
sh, sz, p sh, sz, p
sh, d sh, d sh sh
a
Comprising only the glomeruli located at the anterior antennal lobe of male. Classes refer to the distinctness of glomeruli (for details see Section 2). c Deviation(s) from class distinctness is indicated under comment: sh: shape; sz: size; p: position; d: demarcation. b
In male moths the MGC consists of 2e4 glomeruli positioned close to the entrance of the antennal nerve. Each of these malespecific units receive input from a population of ORNs that detect one particular constituent of female-produced semiochemicals (Hansson et al., 1992; Ochieng et al., 1995; Todd et al., 1995; Berg
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et al., 1998, 2005; Lee et al., 2006a). Thus, the number of macroglomerular sub-structures seems to be correlated with the number of physiological male-specific receptor neuron categories. However, there also are exceptions, e.g. in Helicoverpa zea, ORNs that detect different semiochemicals are reported to project to a single MGC subunit (Lee et al., 2006b). Moreover, in Lobesia botrana and Cydia pomonella (Tortricidea), only one large glomerulus is present in the ALs of males, while three different components serve as pheromones (Masante-Roca et al., 2005; Varela et al., 2009). Also in the cockroach, Periplaneta americana, two types of pheromone receptor neurons tuned to the female-produced attractants periplanone A and periplanone B, respectively, are reported to target one malespecific unit, the MG (Boeckh et al., 1970; Boeckh and Selsam, 1984). The results presented here, demonstrate the presence of a male-specific antennal-lobe structure of H. diomphalia, an MG similar to that of cockroaches. The prominence of the MG in this Coleopteran species, both as concern size and position, indicates the importance of pheromone signals for reproductive behavior. 4.2. Antennal glumeruli of H. diomphalia: number and description The number of approximately 60 antennal-lobe glomeruli in H. diomphalia, as determined for the females in the present study, is in correspondence with previous reports from other insect species. In particular, this number is similar to the finding of ca 70 glomeruli in the red flour beetle T. castaneum (Dreyer et al., 2010). As compared to mammals, which often have thousands of glomeruli in their primary olfactory center (Menco, 1980), insects usually possess a considerably smaller number of typically 50e250 glomeruli (Rospars, 1988; Anton and Homberg, 1999; Schachtner et al., 2005). It seems that the numbers of glomeruli are similar across species of the same insect group. Thus, Diptera generally have numbers ranging from 40 to 50, Lepidoptera 60 to 65, and Hymenoptera more than 100 (Schachtner et al., 2005; Ignell and Hansson, 2005; Ghaninia et al., 2007; Masante-Roca et al., 2005; Varela et al., 2009; Nishikawa et al., 2008; Zube and Rössler, 2008). Although we numbered and named all found glomeruli, one must recognize that it is an estimation. Two factors could affect this estimation. One is that smaller glomeruli may not be visible due to the thickness of the sections causing a smaller estimation. The other is that the unclear boundary of Class 3 glomeruli may increase the estimation. Total of 8 Class 3 glomeruli were identified in females. Unclear boundaries were present between AD3 and AD5, AD6 and AC6, PC5 and PC6, PD2 and PD3. Therefore, it is possible that the pairs we named in the study are actual one glomerulus. These two factors are the likely reasons for the discrepancy of number of glomeruli in the four samples. In males the glomeruli at the posterior part of male ALs were more densely packed and individual glomeruli were difficult to delineate because of the unclear boundary. This matter of fact may of course be due to technical problems concerning penetration of dye in the deeper brain layers. However, the distinctiveness of glomeruli may also be closely related to the composition of the neural network, i.e., the density of neuropil. The number of afferent neurons in posteriorly located glomeruli may for instance be lower than that of more anteriorly positioned glomeruli (Ghaninia et al., 2007). Regardless of the cause, the estimates should be improved by alternative staining methods or novel molecular techniques in order to verify and complement the presented data. Acknowledgments We acknowledge Dr. Bente G. Berg (Norwegian University of Science and Technology) for critically reading and greatly improving the manuscript. We also thank two anonymous reviewers for their
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