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Fine structure of the antennal glands of the ant nest beetle Paussus favieri (Coleoptera, Carabidae, Paussini)
Arthropod Structure & Development 38 (2009) 293–302
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Fine structure of the antennal glands of the ant nest beetle Paussus favieri (Coleoptera, Carabidae, Paussini) Andrea Di Giulio a, *, Marco Valerio Rossi Stacconi b, Roberto Romani b a b
` degli Studi Roma Tre, Viale G. Marconi, 446, 00146 Roma, Italy Dipartimento Biologia, Universita ` degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy Dipartimento di Scienze Agrarie ed Ambientali, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2008 Accepted 19 January 2009
The antennae of the ant nest beetle Paussus favieri are studied by using both SEM and TEM. In the myrmecophilous genus Paussus, these structures are composed of three joints: scape, pedicel and a wide third joint, the ‘‘antennal club’’, resulting from the fusion of antennomeres A3–A11 (flagellum). The antennal club shows an exceptional glandular activity, with the presence of pores mostly crowded in special hairless cuticular areas, surrounding the base of single setae, grouped at the base of tufts of setae, or positioned inside deep pockets that store the secretions, with filiform material arising from them. The surface of A1 and A3 are covered by mechanoreceptors, modified to spread the glandular exudates, while the chemoreceptors are restricted to the apex of the club. The fine structural analysis shows a great number of antennal glands, that can be referred to three main typologies: type A (GhA) bi-cellular, composed of a large secretory cell and a small duct cell, positioned close to the antennal surface; type B (GhB), tri-cellular, composed of two secretory cells and one duct cell, less frequent and positioned deep inside the antennal club; type C (GhC), rare, located deeply within the antennal lumen, in the vicinity of the trophocytes. This complexity indicates that more than one substance could be released from the antennae. Possible functional aspects of the secretions dealing with symbiotic interaction with the host ants are discussed. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Coleoptera Myrmecophily Antennal glands Functional anatomy Sensilla Antennal microstructures
1. Introduction The antennae are considered to be the most important insect sensory organs, mainly used for intraspecific communication and food search. While the sensorial function of the antennae and the variety of antennal sensilla has received great attention in the last fifty years (Altner and Prillinger, 1980; Zacharuk, 1985; Steinbrecht, 1987; Bartlet et al., 1999; Merivee et al., 2001), their glandular function is still scarcely known, and has been studied in detail only in Hymenoptera (Bin and Vinson, 1986; Bin et al., 1989, 1999; Isidoro et al., 1996, 1999, 2000; Romani et al., 2003, 2005, 2006; Sacchetti et al., 1999; Guerrieri et al., 2001) and in some Coleoptera (Yung, 1938; Cammaerts, 1974; Martin, 1975, 1977; Bartlet et al., 1994; De Marzo, 1994; Skilbeck and Anderson, 1994; Weis et al., 1999; Giglio et al., 2005). The presence of antennal glands can be surmised by external inspections of the antennae with the scanning electron microscope (SEM). In this way it is possible to observe pores (openings of
* Corresponding author. Tel.: þ39 0657336323; fax: þ39 0657336321. E-mail addresses: [email protected] (A. Di Giulio), [email protected] (R. Romani). 1467-8039/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2009.01.001
cuticular ducts) of different size, scattered, crowded or surrounding the base of antennal setae, often with filaments of material arising from them. The glandular function can be performed by both sexes or can be restricted to one sex (usually the male), and the number and position of antennal glands can vary among sexes, involving one or few adjacent antennomeres, or the whole antenna. Like other exocrine glands, antennal glands have an epidermal origin and their structure fits the types 1 and 3 of Noirot and Quennedey (1974, 1991) and Quennedey (1998). Recent morphological studies on antennae of Coleoptera, both Adephaga and Polyphaga (Nagel, 1979; Bartlet et al., 1994; De Marzo, 1994; Weis et al., 1999; Giglio et al., 2005; Di Giulio, Personal observation) on various species of Carabidae Paussinae, Scarabaeidae and Meloidae, indicate that antennal glands are widespread in this order, and can be part of the ground-plan of Coleoptera (Weis et al., 1999). The main functions proposed for the antennal exocrine glands are: (1) secretion of pheromones used in species, kin or sex recognition and communication (Bin et al., 1989, 1999; Skilbeck and Anderson, 1994; Isidoro et al., 1996, 1999; Romani et al., 2003, 2005; Turco et al., 2003); (2) host recognition (in parasitic Hymenoptera) (Isidoro et al., 1996, 2001); (3) social integration; (4) protection for antennal sensilla; (5) lubrication for the antennomere joints to reduce friction in moving parts of the cuticle (Cammaerts, 1974;
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Martin, 1977; Skilbeck and Anderson, 1994; Giglio et al., 2005); (6) signaller of spatial information (Strohm and Linsenmair, 1994/95); (7) bacteria cultivation organs (Goettler et al., 2007). Another peculiar function of the antennal glands is that limited to a heterogeneous assemblage of insects that live as symbionts of ants (Ho¨lldobler and Wilson, 1990). In these so-called myrmecophiles, the antennal secretions seem to be of primary importance for the success of the symbiotic interaction with the host ants (Cammaerts, 1974; Kistner, 1982; Ho¨lldobler and Wilson, 1990). In the myrmecophilous tribe Paussini (Carabidae, Paussinae), an increased glandular function at the level of the antennae can even exceed the primary sensorial function, and influences the position and the shape of antennal mechanoreceptors (this work; Di Giulio et al., in preparation). A few available histological studies on myrmecophilous beetles outline the presence of complexes of epidermal secretory glands at the base of ‘‘symphilous organs’’, composed of unicellular (Wasmann, 1903; Yung, 1938; Alpert, 1994), or uni- and tri-cellular glands (respectively types A and B of Cammaerts (1974)). However, the fine structure of the glandular units remained undescribed. In this paper we analyse and describe the external morphology, the fine structure and the distribution of the antennal glands in the Mediterranean ant nest beetle Paussus favieri Fairmaire (Fig. 1), through both scanning electron microscopy (SEM) and transmission electron microscopy (TEM), in order to infer about the relationships between ants and their guests and to contribute to the debate on the evolution of myrmecophily in these insects. 2. Materials and methods 2.1. Material examined Three specimens (one female and two males) of P. favieri from ‘‘Morocco, Tizi-n-Test, 2063 m a.s.l., 5.V.2006 A. Di Giulio and
F. Turco leg.’’, sampled together with 7 more specimens in an ant nest of Pheidole pallidula. 2.2. Scanning electron microscopy One dried male was kept overnight in a detergent water solution, cleaned by ultrasounds for 15 s, rinsed in water, dehydrated by passing through a graded ethanol series, critical point-dried in a Balzer UnionÒ CPD 030 unit, gold coated in an EmitechÒ K550 unit, and finally examined with a PhilipsÒ XL30 microscope (L.I.M.E., ‘‘Roma Tre’’ University, Rome, Italy). 2.3. Transmission electron microscopy For TEM analysis, one male and one female were CO2 anesthetized and immediately immersed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer þ 5% sucrose, pH 7.2–7.3. The antennae were removed from the head, the antennal club was cut transversally and replaced in the fixative to improve penetration, and held there for 2 h at 4 C. They were then rinsed overnight in cacodylate buffer, post-fixed in 1% osmium tetroxide for 1 h at 4 C and rinsed in cacodylate buffer. Samples were dehydrated in a graded ethanol series followed by embedding in Epon–Araldite with propylene oxide as a bridging solvent. Thin sections were cut with a DrukkerÒ diamond knife on an LKBÒ ‘‘Nova’’ ultramicrotome, and mounted on Formvar-coated 50 mesh grids. Sections were examined with a PhilipsÒ EM 208 microscope after staining with uranyl acetate (20 min, room temperature) and lead citrate (5 min, room temperature) (C.U.M.E., Perugia University, Italy). Digital pictures (1280 1024 pixels, 16b, uncompressed greyscale Tiff files) were obtained using a high resolution digital camera ColorView III (SISÒ). 3. Results 3.1. Gross morphology of the antennae The antennae of P. favieri (length about 1.2 mm) are composed of three joints: a cylindrical and slightly elongated scape (A1) (length ¼ 0.32 mm, width ¼ 0.2 mm; Fig. 2A and B), a small and globular pedicel (A2) (width ¼ 50 mm), and the so-called ‘‘antennal club’’ (Fig. 2A and B), resulting from the fusion of antennomeres A3–A11. This last segment is wide, sub-triangular, swollen and strongly asymmetrical, with a broadly rounded apex (length 0.82 mm, width at base 0.6 mm; Fig. 2D). The anterior margin of the antennal club is keeled and slightly concave (Fig. 2B), while the posterior margin appears medially four-toothed (Fig. 2A), and with a conspicuous, pointed basal spur (Figs. 1 and 2A). The dorsal surface of the antenna is strongly convex (Fig. 2A), while the ventral surface appears concave posteriorly and convex anteriorly (Fig. 2B), furrowed in the posterior half by four transverse, deep pockets (length about 140 mm; Figs. 2B and 3D), ending at the tip of each tooth. The surface of the antenna appears completely smooth. 3.2. Antennal microstructures
Fig. 1. Paussus favieri, dorsal habitus (from Casale et al., 1982).
3.2.1. Sensilla Many modified sensilla chaetica (flattened, falcate, pennate, fringed and/or branched apically) are present on the scape and on the antennal club, singly or arranged in rows (Fig. 2A and C). Two tufts (one dorsal and one ventral) of sensilla chaetica (‘‘antennal symphilous organs’’ or ‘‘bristle tufts’’) (Fig. 3A and B) of simple trichoid shape (often slightly branched at apex), are present subapically on the basal spur. Sensilla basiconica (of various types),
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Fig. 2. Paussus favieri, SEM micrographs of: A: Left antenna, dorsal view. B: Left antenna, ventral view. C: Details of antennomere I (scape), showing a modified seta and glandular pores. D: Antennal apex, anteroventral view. Scale bars: A and B ¼ 200 mm; C ¼ 10 mm; D ¼ 100 mm.
coeloconica and campaniformia are present at the apex of the antennal club, grouped in a semicircular sensorial area (Fig. 2D). 3.2.2. Pores On the scape and the antennal club, a conspicuous presence of small scattered pores (0.2–0.3 mm) is evident. These pores are also present on the apical sensorial area and along the antennal margin. The base of the modified sensilla chaetica is often encircled by 2–4 pores (0.25 mm). Additionally, the antennal club shows a medioventral, multiperforate area (Fig. 3C) close to the base; each pore of this area (size 1–1.5 mm) appears positioned in the middle of a round, flattened depression. Many pores of two different sizes (0.25–0.3 and 0.8–1 mm) are also present at the base of the setae that compose the antennal symphilous organs (Fig. 3B) and inside the ventral pockets (1.25 mm, Fig. 3D). Abundant filiform material arises from most pores, particularly from those of the ventral pockets and of the symphilous organs (Fig. 3B and D).
3.3. Glandular structures Light microscopic investigations show that the antennal lumen contains a relative low number of cells and a massive presence of electron-lucid material in the form of sub-spherical bodies (about 30% of the entire internal space of the antennomere) (Fig. 4A). Fine structural observations revealed that these bodies (diameter ranging from 2 to 15 mm) are actually lipid droplets embedded
within the cytoplasm of trophocytes (Fig. 6C and D). The subcuticular region of the antennal wall is occupied by a dense, evenly distributed glandular epithelium. TEM investigations revealed the presence of two main types of glandular structures and a third type occurring in low number (Fig. 4A). All types belong to Class 3 secretory cells, being characterized by the presence of secretory cells and duct cells (Noirot and Quennedey, 1974; Quennedey, 1998).
3.4. Type A glands Type A glands (GhA) are the most abundant and are distributed on almost the entire sub-cuticular region of the antennal wall, with a higher concentration on the ventral side. Each glandular unit is composed of two cells: a large inner cell involved in the secretory activity and a small outer cell producing the cuticular evacuating duct (Fig. 4B). The secretory cell shows a big, rounded nucleus and a cytoplasm with a remarkable presence of mitochondria and a smooth endoplasmic reticulum. Secretory products were not found in the investigated specimens (Fig. 4E and F). The receiving canal is wide and highly microvillate, and occupies almost half of the cell volume (Fig. 4B). It presents externally a discontinuous epicuticular layer, delimiting an internal space of 5 mm (length) and 2–3 mm (width). At this level, the receiving canal is completely surrounded by microvilli that show, in cross-section, numerous microfilaments (Fig. 4D). The receiving canal lumen is characterized
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Fig. 3. Paussus favieri, SEM micrographs of: A: Basal spur of antennal club, dorsal tuft. B: Detail of ventral tuft, showing glandular pores and their secretions. C: Medioventral, multiperforate area with filaments of secretions. D: Ventral pocket of antennal club with pores of various sizes and their secretions. Scale bars: A ¼ 50 mm; B and D ¼ 10 mm; C ¼ 20 mm.
by a microfibrillar structure made of epicuticular filaments for the carriage of the secretion, therefore, giving a ‘‘spongy’’ appearance to the structure (Fig. 4C). Proximally, the evacuating duct extends deeply into the receiving canal, while distally it is surrounded by a duct cell. The evacuating duct shows a peculiar ‘‘trachea-like’’ structure, given the presence of cuticular reinforcements resembling the taenidia (Fig. 4C). The duct cell is smaller than the secretory cell, and often appears flattened against the cuticular wall. The cytoplasm of the duct cell is reduced, showing few organelles.
a discontinuous activity of the cells (Fig. 5B–D). The apical region of the cell houses a large and convoluted receiving canal, surrounded by packed microvilli. The receiving canal is made up of a single layer of epicuticle (thickness about 0.2 mm) which the microvilli contact. Along the receiving canal several protrusions occur (Fig. 5A and B). At its distal part, the receiving canal continues into the outer secretory cell, where a second receiving canal occurs (Fig. 5B). However, in all the specimens investigated we never observed secretory vesicles in the outer secretory cell. The electron-dense secretion is released at the level of the pockets, where the evacuating ducts connect with the external pores.
3.5. Type B glands 3.6. Type C glands Type B glands (GhB) are less abundant than GhA and are located inside the antennal lumen, therefore there is no direct contact between these glands and the antennal wall (Fig. 4A). However these glandular units are concentrated in the vicinity of the pockets, where secretory products are released. Fine structural observations revealed that each gland is made up of at least three cells: two secretory cells and one duct cell (Fig. 5A and B). The cytoplasm of the inner secretory cell is characterized by the presence of an abundant rough endoplasmic reticulum and well-developed Golgi apparati (Fig. 5E and F). Large clusters of electron-dense secretory vesicles (diameter ranging from 0.3 to 1 mm) are often observed, although in some cases there are few or no vesicles, possibly due to
A third type of glandular structures, defined as Type C glands was observed. GhC is located deeply within the antennal lumen, in the vicinity of the trophocytes. These glands are characterized by a large secretory cell (diameter about 50 mm), the cytoplasm of which is very rich in secretory vesicles of different electron densities (Fig. 6A and B). Electron-dense vesicles surround the receiving canal, which shows numerous microvilli oriented towards the central lumen. Because of the rarity of these secretory cells in the sections that are available, we were unable to determine the number of secretory cells and the locations of the external openings. Thus it was not possible to give a detailed descriptions of GhC.
Fig. 4. Antennal glands in Paussus favieri. A: Light micrograph showing, in cross-section, the distribution of glands type A (GhA), type B (GhB) and trophocytes (TR) within the antennal club. B–F: TEM pictures of GhA. B: Longitudinal section of a GhA unit showing the secretory cell (SC) with the receiving canal (RC), and the duct cell (DC) with the evacuating duct (ED) entering the antennal cuticle (CU). C: Details of the receiving canal and the proximal part of the evacuating duct. The receiving canal is bordered by microvilli (MV) and presents tiny pores (arrowheads) and numerous epicuticular filaments (EF) for the carriage of the secretion. The proximal part of the evacuating duct shows a peculiar ‘‘trachea-like’’ structure. D: Close up view of the microvilli bordering the receiving canal. Numerous microfilaments (MF) can be observed in cross-section. E–F: Details of the secretory cell with the large rounded nucleus (N), smooth endoplasmic reticulum (SER) and mitochondria (MT). Scale bars: A ¼ 100 mm; B ¼ 5 mm; C and F ¼ 1 mm; D ¼ 0.5 mm; E ¼ 2 mm.
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Fig. 5. Antennal glands type B (GhB) in Paussus favieri. A and B: TEM longitudinal sections of GhB showing the inner (ISC) and outer (OSC) secretory cells. In A the inner secretory cell presents a large receiving canal bordered by microvilli (MV) where clumps of electron-dense secretion accumulate. In B the receiving canal (RC) is lighter, while the cytoplasm shows a large number of dark secretory vesicles. The outer secretory cell shows basally a receiving canal. C–E: TEM sections of the inner secretory cell showing the receiving canal either filled with secretion (S) (in C) or empty (in D). E–F: large stacks of rough endoplasmic reticulum (RER) arranged in parallel and Golgi apparatus (GO) of the inner secretory cell. IN, basal infoldings; MT, mitochondria; N, nucleus; SJ, septate junctions. Scale bars: A ¼ 5 mm; B–D ¼ 2 mm; E ¼ 1 mm; F ¼ 0.5 mm.
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3.7. Bristle tufts Thin sections of the proximal part of the club show that the bristles which compose the lateral tufts of the spur are modified setae, showing a smooth, aporous external wall with an irregular section. The cuticular shaft is made of thick, continuous cuticle, leaving a small, hollow central lumen. Each bristle is inserted in the antennal wall through a socket, where it is suspended by means of a joint membrane (Fig. 7A and B). At this level, the single neuron innervating these sensilla ends in a tubular body (Fig. 7B). After the ciliary region, the outer dendritic segment of each neuron is enclosed by the dendrite sheath produced by the thecogen cell (Fig. 7D). 4. Discussion Myrmecophily is a wide phenomenon that has been reported from 29 extant insect orders, with the majority of diversity found in five orders: Hemiptera, Coleoptera, Diptera, Lepidoptera and Hymenoptera (Kistner, 1982; Ho¨lldobler and Wilson, 1990). Since the communication and the social integration between ants is mainly chemical (Ho¨lldobler and Wilson, 1990), the myrmecophiles, to be accepted and to survive inside the host colonies,
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produce attractive substances that mime the ant’s chemical key signals, or appease their hosts (Nagel, 1979, 1997; Ho¨lldobler and Wilson, 1990; Geiselhardt et al., 2007), rendering ‘‘invisible’’ or welcome these odd guests. For this reason, most symphilous myrmecophiles increase their exocrine glandular activity, if compared to their non-symbiont relatives. The chemicals can be secreted by different parts of the body, including the antennae (Yung, 1938; Wasmann, 1903; Cammaerts, 1974), that are the first organs that contact the host specimens inside the galleries of the nests. In the genus Paussus and a few other genera of Paussini, the whole flagellum (basically composed of 9 antennomeres) is fused into one single ‘‘antennal club’’, sometimes with remnants of serial subdivision. This antennal club shows in Paussus a great structural heterogeneity and can often assume bizarre shapes (flattened, enlarged, lenticular, globular, concave, elongate, etc.) (Darlington, 1950; Nagel, 1979; Luna de Carvalho, 1989). The reason of this morphological diversity is still unknown, but it is at least partially connected to the amplified glandular function of the antennae and to different ways to spread, offer and store the antennal exudates. The present morphological study, that follows other pioneer histological studies on Paussini (Wasmann, 1903; Yung, 1938), shows that the antennae of these myrmecophilous beetles have an
Fig. 6. A and B: TEM cross-sections of antennal glands type C (GhC) in Paussus favieri. These cells show a large receiving canal (RC) and a large amount of secretory vesicles of different electron densities. C and D: Trophocytes with large lipidic droplets (LD) enclosed within the cytoplasm. GhB, antennal glands type B; N, nucleus, MT, mitochondria. Scale bars: A and B ¼ 10 mm; C ¼ 5 mm; D ¼ 2 mm.
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Fig. 7. Antennal hair tufts in Paussus favieri: A: SEM micrograph showing a fractured basal part of the dorsal hair tuft, with details of the hair sockets. B–D: TEM micrographs of the single sensory neuron associated with each hair bristle. In B the distal end of the outer dendritic segment enclosed by the dendrite sheath (DS) differentiate a tubular body (TB) which stops at the level of the joint membrane (JM). In C a longitudinal section of the sensory neuron (SN) between adjacent antennal glands type A (GhA). In D details of a longitudinal section taken at the ciliary region level. CC, ciliary collar; CR, ciliary rootlets; CU, cuticle; IDS, inner dendritic segment; ODS, outer dendritic segment. Scale bars: A and B ¼ 10 mm; C ¼ 5 mm; D ¼ 2 mm.
exceptional glandular activity. In P. favieri, and probably in all species of Paussini (Nagel, 1979; Di Giulio et al., unpublished data), this is observable from the external by the presence of a great number of pores, different in structure and position, spread throughout the antenna, surrounding the base of single modified setae, crowded in special tegumental areas, grouped at the base of tufts of hairs, or crowded inside deep pockets that store the secretions, with filiform material similar to a paste arising from them. The ultrastructural analysis confirms the complexity of the antennal glandular apparatus of P. favieri, with different types of exocrine glands localized in different regions of the antenna, or mixed without a clear connection to a specific area. Glands type A (GhA) are widespread under the antennal cuticle, mainly concentrated on the ventral side and on symphilous organs. Glands Type B (GhB) deeper within the antennal lumen, particularly in the region close to the antennal pockets. Glands Type C (GhC) have been found deep in the antennal lumen, close to the area housing trophocytes. The general structure of the different
gland types refers to Class 3 secretory cells, according to the classification of insect epidermal glands proposed by Noirot and Quennedey (1974) and Quennedey (1998). In fact, in all cases (GhC are located) each glandular unit is composed of one (GhA) or two (GhB) secretory cells and a duct cell. The secretion produced by the secretory cells is then carried by a cuticular evacuating duct and released through external pores. The occurrence of antennal glands has been reported in several families of coleopterans (Yung, 1938; Matthes, 1970; Cammaerts, 1974; Martin, 1975). Furthermore, the studies carried out using TEM techniques have revealed that antennal glands in Coleoptera belong either to Class 1 (Skilbeck and Anderson, 1994) or Class 3 (Bartlet et al., 1994; Giglio et al., 2005). However, in P. favieri there is an exceptional development of antennal glandular structures in terms of glandular types and their abundance. Despite the large number of secretory cells, we did not find any structure related to the storage of the secretion. However, we can hypothesize that the large receiving canal of GhA could serve as temporary storage for the secretion. The ultrastructural
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features of the secretory cells, i.e. the presence of rough endoplasmic reticulum associated with Golgi apparatus, and the abundance of electron-dense vesicles (at least in GhB) strongly suggest a proteinaceous nature for the secretion. The absence of secretory products within the cytoplasm of GhA secretory cells could be interpreted as a discontinuous activity as regards the secretion, and this was found for the secretory cells of GhB as well. The abundance of the secretion on the antennal surface in all the investigated specimens strongly supports a low or absent volatility for the secretory products. Moreover, the presence of trophocytes with inclusions of large fat vesicles could be interpreted as a storage to supply the energy for the high metabolic activities of the gland cells. It is worth to note that the modified setae present on antennal surface and mostly those that compose the tufts of the symphilous organs are functional mechanoreceptors. Actually, most previous authors working on Paussini and other myrmecophiles used the term ‘‘trichomes’’ for such structures (see Geiselhardt et al., 2007 and references therein). Though not always explicitly defined, this term would refer to uninnervated hairs only functioning in dispensing chemical secretions from underlying glands. Since no muscles have been found associated with the different glands, a feedback control system for the release of the secretions, involving also the mechanosensory hairs, can be hypothesized. The substances produced by the antennal glands of Paussini do not exclusively consist of fat and volatile chemicals (e.g. ethereal oils), like for example in some myrmecophilous Staphilinidae, but represent solid fine ‘‘foods’’ of unknown chemical composition (Nagel, 1979). These exudates are often spread by modified setae, singly or grouped into tufts actively liked by the ants in a ‘‘friendly’’ way (Nagel, 1979; Geiselhardt et al., 2007). Similar tufts of setae and exocrine glands are also present in other parts of the body like frons, prothorax, elytra and pygidium. The substances are thought to contain attractive allomones probably important to avoid aggressions and to induce the host to accept them inside the nests (Geiselhardt et al., 2007). Thus, the specific function (appeasment, adoption, chemical mimicry, food source, etc.) remains speculative or anecdotical (see Geiselhardt et al., 2007, for a review). The complexity and the variety of the antennal glands of P. favieri lead us to the hypothesis that more than one substance can be produced by different areas of the same antenna (also testified by the different electron densities of the secretory product). It is also possible that different glandular complexes can be active in different moments of the symbiotic interaction. Though no significant differences have been observed between males and females in the number and structure of antennal glands, a function for the intersexual communication cannot be excluded, besides the acknowledged myrmecophilous function. Acknowledgments We are grateful to Federica Turco (Queensland Museum, Brisbane, Australia) for her precious help in field collection and rearing of the specimens of Paussus favieri studied in this work. We also thank Dorotea De Propris (Rome, Italy) for her assistance in SEM preparation and observations, and the discussions on paussine antennal microstructures. TEM data were obtained at the CUME, Perugia University (Italy), SEM data at LIME of ‘‘Roma Tre’’ University (Rome, Italy). We thank Augusto Vigna Taglianti (University ‘‘La Sapienza’’ Rome, Italy) and Achille Casale (University of Sassari, Sassari, Italy) for their permission to publish the drawing of P. favieri (Fig. 1) and Wendy Moore (University of Arizona, Tucson, USA) for the linguistic revision of the manuscript.
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Die Fa¨chelbalz von Cercoma scha Verhandlungen der Deutschen Zoologischen Gesellschaft 63, 316–322. Merivee, E., Ploomi, A., Luik, A., Rahi, M., Sammelselg, V., 2001. Antennal sensilla of the ground beetle Platynus dorsalis (Pontoppidan, 1763) (Coleoptera, Carabidae). Microscopy Research and Technique 55, 339–349. Nagel, P., 1979. Aspects of the evolution of myrmecophilous adaptations in Paussinae (Coleoptera: Carabidae). Miscellaneous Papers of Agricultural University Wageningen 18, 15–34. Nagel, P., 1997. New fossil paussids from Dominican amber with notes on the phylogenetic systematics of the paussine complex (Coleoptera: Carabidae). Systematic Entomology 22, 345–362.
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Fine structure of the antennal glands of the ant nest beetle Paussus favieri (Coleoptera, Carabidae, Paussini) Andrea Di Giulio a, *, Marco Valerio Rossi Stacconi b, Roberto Romani b a b
` degli Studi Roma Tre, Viale G. Marconi, 446, 00146 Roma, Italy Dipartimento Biologia, Universita ` degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy Dipartimento di Scienze Agrarie ed Ambientali, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2008 Accepted 19 January 2009
The antennae of the ant nest beetle Paussus favieri are studied by using both SEM and TEM. In the myrmecophilous genus Paussus, these structures are composed of three joints: scape, pedicel and a wide third joint, the ‘‘antennal club’’, resulting from the fusion of antennomeres A3–A11 (flagellum). The antennal club shows an exceptional glandular activity, with the presence of pores mostly crowded in special hairless cuticular areas, surrounding the base of single setae, grouped at the base of tufts of setae, or positioned inside deep pockets that store the secretions, with filiform material arising from them. The surface of A1 and A3 are covered by mechanoreceptors, modified to spread the glandular exudates, while the chemoreceptors are restricted to the apex of the club. The fine structural analysis shows a great number of antennal glands, that can be referred to three main typologies: type A (GhA) bi-cellular, composed of a large secretory cell and a small duct cell, positioned close to the antennal surface; type B (GhB), tri-cellular, composed of two secretory cells and one duct cell, less frequent and positioned deep inside the antennal club; type C (GhC), rare, located deeply within the antennal lumen, in the vicinity of the trophocytes. This complexity indicates that more than one substance could be released from the antennae. Possible functional aspects of the secretions dealing with symbiotic interaction with the host ants are discussed. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Coleoptera Myrmecophily Antennal glands Functional anatomy Sensilla Antennal microstructures
1. Introduction The antennae are considered to be the most important insect sensory organs, mainly used for intraspecific communication and food search. While the sensorial function of the antennae and the variety of antennal sensilla has received great attention in the last fifty years (Altner and Prillinger, 1980; Zacharuk, 1985; Steinbrecht, 1987; Bartlet et al., 1999; Merivee et al., 2001), their glandular function is still scarcely known, and has been studied in detail only in Hymenoptera (Bin and Vinson, 1986; Bin et al., 1989, 1999; Isidoro et al., 1996, 1999, 2000; Romani et al., 2003, 2005, 2006; Sacchetti et al., 1999; Guerrieri et al., 2001) and in some Coleoptera (Yung, 1938; Cammaerts, 1974; Martin, 1975, 1977; Bartlet et al., 1994; De Marzo, 1994; Skilbeck and Anderson, 1994; Weis et al., 1999; Giglio et al., 2005). The presence of antennal glands can be surmised by external inspections of the antennae with the scanning electron microscope (SEM). In this way it is possible to observe pores (openings of
* Corresponding author. Tel.: þ39 0657336323; fax: þ39 0657336321. E-mail addresses: [email protected] (A. Di Giulio), [email protected] (R. Romani). 1467-8039/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2009.01.001
cuticular ducts) of different size, scattered, crowded or surrounding the base of antennal setae, often with filaments of material arising from them. The glandular function can be performed by both sexes or can be restricted to one sex (usually the male), and the number and position of antennal glands can vary among sexes, involving one or few adjacent antennomeres, or the whole antenna. Like other exocrine glands, antennal glands have an epidermal origin and their structure fits the types 1 and 3 of Noirot and Quennedey (1974, 1991) and Quennedey (1998). Recent morphological studies on antennae of Coleoptera, both Adephaga and Polyphaga (Nagel, 1979; Bartlet et al., 1994; De Marzo, 1994; Weis et al., 1999; Giglio et al., 2005; Di Giulio, Personal observation) on various species of Carabidae Paussinae, Scarabaeidae and Meloidae, indicate that antennal glands are widespread in this order, and can be part of the ground-plan of Coleoptera (Weis et al., 1999). The main functions proposed for the antennal exocrine glands are: (1) secretion of pheromones used in species, kin or sex recognition and communication (Bin et al., 1989, 1999; Skilbeck and Anderson, 1994; Isidoro et al., 1996, 1999; Romani et al., 2003, 2005; Turco et al., 2003); (2) host recognition (in parasitic Hymenoptera) (Isidoro et al., 1996, 2001); (3) social integration; (4) protection for antennal sensilla; (5) lubrication for the antennomere joints to reduce friction in moving parts of the cuticle (Cammaerts, 1974;
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Martin, 1977; Skilbeck and Anderson, 1994; Giglio et al., 2005); (6) signaller of spatial information (Strohm and Linsenmair, 1994/95); (7) bacteria cultivation organs (Goettler et al., 2007). Another peculiar function of the antennal glands is that limited to a heterogeneous assemblage of insects that live as symbionts of ants (Ho¨lldobler and Wilson, 1990). In these so-called myrmecophiles, the antennal secretions seem to be of primary importance for the success of the symbiotic interaction with the host ants (Cammaerts, 1974; Kistner, 1982; Ho¨lldobler and Wilson, 1990). In the myrmecophilous tribe Paussini (Carabidae, Paussinae), an increased glandular function at the level of the antennae can even exceed the primary sensorial function, and influences the position and the shape of antennal mechanoreceptors (this work; Di Giulio et al., in preparation). A few available histological studies on myrmecophilous beetles outline the presence of complexes of epidermal secretory glands at the base of ‘‘symphilous organs’’, composed of unicellular (Wasmann, 1903; Yung, 1938; Alpert, 1994), or uni- and tri-cellular glands (respectively types A and B of Cammaerts (1974)). However, the fine structure of the glandular units remained undescribed. In this paper we analyse and describe the external morphology, the fine structure and the distribution of the antennal glands in the Mediterranean ant nest beetle Paussus favieri Fairmaire (Fig. 1), through both scanning electron microscopy (SEM) and transmission electron microscopy (TEM), in order to infer about the relationships between ants and their guests and to contribute to the debate on the evolution of myrmecophily in these insects. 2. Materials and methods 2.1. Material examined Three specimens (one female and two males) of P. favieri from ‘‘Morocco, Tizi-n-Test, 2063 m a.s.l., 5.V.2006 A. Di Giulio and
F. Turco leg.’’, sampled together with 7 more specimens in an ant nest of Pheidole pallidula. 2.2. Scanning electron microscopy One dried male was kept overnight in a detergent water solution, cleaned by ultrasounds for 15 s, rinsed in water, dehydrated by passing through a graded ethanol series, critical point-dried in a Balzer UnionÒ CPD 030 unit, gold coated in an EmitechÒ K550 unit, and finally examined with a PhilipsÒ XL30 microscope (L.I.M.E., ‘‘Roma Tre’’ University, Rome, Italy). 2.3. Transmission electron microscopy For TEM analysis, one male and one female were CO2 anesthetized and immediately immersed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer þ 5% sucrose, pH 7.2–7.3. The antennae were removed from the head, the antennal club was cut transversally and replaced in the fixative to improve penetration, and held there for 2 h at 4 C. They were then rinsed overnight in cacodylate buffer, post-fixed in 1% osmium tetroxide for 1 h at 4 C and rinsed in cacodylate buffer. Samples were dehydrated in a graded ethanol series followed by embedding in Epon–Araldite with propylene oxide as a bridging solvent. Thin sections were cut with a DrukkerÒ diamond knife on an LKBÒ ‘‘Nova’’ ultramicrotome, and mounted on Formvar-coated 50 mesh grids. Sections were examined with a PhilipsÒ EM 208 microscope after staining with uranyl acetate (20 min, room temperature) and lead citrate (5 min, room temperature) (C.U.M.E., Perugia University, Italy). Digital pictures (1280 1024 pixels, 16b, uncompressed greyscale Tiff files) were obtained using a high resolution digital camera ColorView III (SISÒ). 3. Results 3.1. Gross morphology of the antennae The antennae of P. favieri (length about 1.2 mm) are composed of three joints: a cylindrical and slightly elongated scape (A1) (length ¼ 0.32 mm, width ¼ 0.2 mm; Fig. 2A and B), a small and globular pedicel (A2) (width ¼ 50 mm), and the so-called ‘‘antennal club’’ (Fig. 2A and B), resulting from the fusion of antennomeres A3–A11. This last segment is wide, sub-triangular, swollen and strongly asymmetrical, with a broadly rounded apex (length 0.82 mm, width at base 0.6 mm; Fig. 2D). The anterior margin of the antennal club is keeled and slightly concave (Fig. 2B), while the posterior margin appears medially four-toothed (Fig. 2A), and with a conspicuous, pointed basal spur (Figs. 1 and 2A). The dorsal surface of the antenna is strongly convex (Fig. 2A), while the ventral surface appears concave posteriorly and convex anteriorly (Fig. 2B), furrowed in the posterior half by four transverse, deep pockets (length about 140 mm; Figs. 2B and 3D), ending at the tip of each tooth. The surface of the antenna appears completely smooth. 3.2. Antennal microstructures
Fig. 1. Paussus favieri, dorsal habitus (from Casale et al., 1982).
3.2.1. Sensilla Many modified sensilla chaetica (flattened, falcate, pennate, fringed and/or branched apically) are present on the scape and on the antennal club, singly or arranged in rows (Fig. 2A and C). Two tufts (one dorsal and one ventral) of sensilla chaetica (‘‘antennal symphilous organs’’ or ‘‘bristle tufts’’) (Fig. 3A and B) of simple trichoid shape (often slightly branched at apex), are present subapically on the basal spur. Sensilla basiconica (of various types),
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Fig. 2. Paussus favieri, SEM micrographs of: A: Left antenna, dorsal view. B: Left antenna, ventral view. C: Details of antennomere I (scape), showing a modified seta and glandular pores. D: Antennal apex, anteroventral view. Scale bars: A and B ¼ 200 mm; C ¼ 10 mm; D ¼ 100 mm.
coeloconica and campaniformia are present at the apex of the antennal club, grouped in a semicircular sensorial area (Fig. 2D). 3.2.2. Pores On the scape and the antennal club, a conspicuous presence of small scattered pores (0.2–0.3 mm) is evident. These pores are also present on the apical sensorial area and along the antennal margin. The base of the modified sensilla chaetica is often encircled by 2–4 pores (0.25 mm). Additionally, the antennal club shows a medioventral, multiperforate area (Fig. 3C) close to the base; each pore of this area (size 1–1.5 mm) appears positioned in the middle of a round, flattened depression. Many pores of two different sizes (0.25–0.3 and 0.8–1 mm) are also present at the base of the setae that compose the antennal symphilous organs (Fig. 3B) and inside the ventral pockets (1.25 mm, Fig. 3D). Abundant filiform material arises from most pores, particularly from those of the ventral pockets and of the symphilous organs (Fig. 3B and D).
3.3. Glandular structures Light microscopic investigations show that the antennal lumen contains a relative low number of cells and a massive presence of electron-lucid material in the form of sub-spherical bodies (about 30% of the entire internal space of the antennomere) (Fig. 4A). Fine structural observations revealed that these bodies (diameter ranging from 2 to 15 mm) are actually lipid droplets embedded
within the cytoplasm of trophocytes (Fig. 6C and D). The subcuticular region of the antennal wall is occupied by a dense, evenly distributed glandular epithelium. TEM investigations revealed the presence of two main types of glandular structures and a third type occurring in low number (Fig. 4A). All types belong to Class 3 secretory cells, being characterized by the presence of secretory cells and duct cells (Noirot and Quennedey, 1974; Quennedey, 1998).
3.4. Type A glands Type A glands (GhA) are the most abundant and are distributed on almost the entire sub-cuticular region of the antennal wall, with a higher concentration on the ventral side. Each glandular unit is composed of two cells: a large inner cell involved in the secretory activity and a small outer cell producing the cuticular evacuating duct (Fig. 4B). The secretory cell shows a big, rounded nucleus and a cytoplasm with a remarkable presence of mitochondria and a smooth endoplasmic reticulum. Secretory products were not found in the investigated specimens (Fig. 4E and F). The receiving canal is wide and highly microvillate, and occupies almost half of the cell volume (Fig. 4B). It presents externally a discontinuous epicuticular layer, delimiting an internal space of 5 mm (length) and 2–3 mm (width). At this level, the receiving canal is completely surrounded by microvilli that show, in cross-section, numerous microfilaments (Fig. 4D). The receiving canal lumen is characterized
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Fig. 3. Paussus favieri, SEM micrographs of: A: Basal spur of antennal club, dorsal tuft. B: Detail of ventral tuft, showing glandular pores and their secretions. C: Medioventral, multiperforate area with filaments of secretions. D: Ventral pocket of antennal club with pores of various sizes and their secretions. Scale bars: A ¼ 50 mm; B and D ¼ 10 mm; C ¼ 20 mm.
by a microfibrillar structure made of epicuticular filaments for the carriage of the secretion, therefore, giving a ‘‘spongy’’ appearance to the structure (Fig. 4C). Proximally, the evacuating duct extends deeply into the receiving canal, while distally it is surrounded by a duct cell. The evacuating duct shows a peculiar ‘‘trachea-like’’ structure, given the presence of cuticular reinforcements resembling the taenidia (Fig. 4C). The duct cell is smaller than the secretory cell, and often appears flattened against the cuticular wall. The cytoplasm of the duct cell is reduced, showing few organelles.
a discontinuous activity of the cells (Fig. 5B–D). The apical region of the cell houses a large and convoluted receiving canal, surrounded by packed microvilli. The receiving canal is made up of a single layer of epicuticle (thickness about 0.2 mm) which the microvilli contact. Along the receiving canal several protrusions occur (Fig. 5A and B). At its distal part, the receiving canal continues into the outer secretory cell, where a second receiving canal occurs (Fig. 5B). However, in all the specimens investigated we never observed secretory vesicles in the outer secretory cell. The electron-dense secretion is released at the level of the pockets, where the evacuating ducts connect with the external pores.
3.5. Type B glands 3.6. Type C glands Type B glands (GhB) are less abundant than GhA and are located inside the antennal lumen, therefore there is no direct contact between these glands and the antennal wall (Fig. 4A). However these glandular units are concentrated in the vicinity of the pockets, where secretory products are released. Fine structural observations revealed that each gland is made up of at least three cells: two secretory cells and one duct cell (Fig. 5A and B). The cytoplasm of the inner secretory cell is characterized by the presence of an abundant rough endoplasmic reticulum and well-developed Golgi apparati (Fig. 5E and F). Large clusters of electron-dense secretory vesicles (diameter ranging from 0.3 to 1 mm) are often observed, although in some cases there are few or no vesicles, possibly due to
A third type of glandular structures, defined as Type C glands was observed. GhC is located deeply within the antennal lumen, in the vicinity of the trophocytes. These glands are characterized by a large secretory cell (diameter about 50 mm), the cytoplasm of which is very rich in secretory vesicles of different electron densities (Fig. 6A and B). Electron-dense vesicles surround the receiving canal, which shows numerous microvilli oriented towards the central lumen. Because of the rarity of these secretory cells in the sections that are available, we were unable to determine the number of secretory cells and the locations of the external openings. Thus it was not possible to give a detailed descriptions of GhC.
Fig. 4. Antennal glands in Paussus favieri. A: Light micrograph showing, in cross-section, the distribution of glands type A (GhA), type B (GhB) and trophocytes (TR) within the antennal club. B–F: TEM pictures of GhA. B: Longitudinal section of a GhA unit showing the secretory cell (SC) with the receiving canal (RC), and the duct cell (DC) with the evacuating duct (ED) entering the antennal cuticle (CU). C: Details of the receiving canal and the proximal part of the evacuating duct. The receiving canal is bordered by microvilli (MV) and presents tiny pores (arrowheads) and numerous epicuticular filaments (EF) for the carriage of the secretion. The proximal part of the evacuating duct shows a peculiar ‘‘trachea-like’’ structure. D: Close up view of the microvilli bordering the receiving canal. Numerous microfilaments (MF) can be observed in cross-section. E–F: Details of the secretory cell with the large rounded nucleus (N), smooth endoplasmic reticulum (SER) and mitochondria (MT). Scale bars: A ¼ 100 mm; B ¼ 5 mm; C and F ¼ 1 mm; D ¼ 0.5 mm; E ¼ 2 mm.
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Fig. 5. Antennal glands type B (GhB) in Paussus favieri. A and B: TEM longitudinal sections of GhB showing the inner (ISC) and outer (OSC) secretory cells. In A the inner secretory cell presents a large receiving canal bordered by microvilli (MV) where clumps of electron-dense secretion accumulate. In B the receiving canal (RC) is lighter, while the cytoplasm shows a large number of dark secretory vesicles. The outer secretory cell shows basally a receiving canal. C–E: TEM sections of the inner secretory cell showing the receiving canal either filled with secretion (S) (in C) or empty (in D). E–F: large stacks of rough endoplasmic reticulum (RER) arranged in parallel and Golgi apparatus (GO) of the inner secretory cell. IN, basal infoldings; MT, mitochondria; N, nucleus; SJ, septate junctions. Scale bars: A ¼ 5 mm; B–D ¼ 2 mm; E ¼ 1 mm; F ¼ 0.5 mm.
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3.7. Bristle tufts Thin sections of the proximal part of the club show that the bristles which compose the lateral tufts of the spur are modified setae, showing a smooth, aporous external wall with an irregular section. The cuticular shaft is made of thick, continuous cuticle, leaving a small, hollow central lumen. Each bristle is inserted in the antennal wall through a socket, where it is suspended by means of a joint membrane (Fig. 7A and B). At this level, the single neuron innervating these sensilla ends in a tubular body (Fig. 7B). After the ciliary region, the outer dendritic segment of each neuron is enclosed by the dendrite sheath produced by the thecogen cell (Fig. 7D). 4. Discussion Myrmecophily is a wide phenomenon that has been reported from 29 extant insect orders, with the majority of diversity found in five orders: Hemiptera, Coleoptera, Diptera, Lepidoptera and Hymenoptera (Kistner, 1982; Ho¨lldobler and Wilson, 1990). Since the communication and the social integration between ants is mainly chemical (Ho¨lldobler and Wilson, 1990), the myrmecophiles, to be accepted and to survive inside the host colonies,
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produce attractive substances that mime the ant’s chemical key signals, or appease their hosts (Nagel, 1979, 1997; Ho¨lldobler and Wilson, 1990; Geiselhardt et al., 2007), rendering ‘‘invisible’’ or welcome these odd guests. For this reason, most symphilous myrmecophiles increase their exocrine glandular activity, if compared to their non-symbiont relatives. The chemicals can be secreted by different parts of the body, including the antennae (Yung, 1938; Wasmann, 1903; Cammaerts, 1974), that are the first organs that contact the host specimens inside the galleries of the nests. In the genus Paussus and a few other genera of Paussini, the whole flagellum (basically composed of 9 antennomeres) is fused into one single ‘‘antennal club’’, sometimes with remnants of serial subdivision. This antennal club shows in Paussus a great structural heterogeneity and can often assume bizarre shapes (flattened, enlarged, lenticular, globular, concave, elongate, etc.) (Darlington, 1950; Nagel, 1979; Luna de Carvalho, 1989). The reason of this morphological diversity is still unknown, but it is at least partially connected to the amplified glandular function of the antennae and to different ways to spread, offer and store the antennal exudates. The present morphological study, that follows other pioneer histological studies on Paussini (Wasmann, 1903; Yung, 1938), shows that the antennae of these myrmecophilous beetles have an
Fig. 6. A and B: TEM cross-sections of antennal glands type C (GhC) in Paussus favieri. These cells show a large receiving canal (RC) and a large amount of secretory vesicles of different electron densities. C and D: Trophocytes with large lipidic droplets (LD) enclosed within the cytoplasm. GhB, antennal glands type B; N, nucleus, MT, mitochondria. Scale bars: A and B ¼ 10 mm; C ¼ 5 mm; D ¼ 2 mm.
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Fig. 7. Antennal hair tufts in Paussus favieri: A: SEM micrograph showing a fractured basal part of the dorsal hair tuft, with details of the hair sockets. B–D: TEM micrographs of the single sensory neuron associated with each hair bristle. In B the distal end of the outer dendritic segment enclosed by the dendrite sheath (DS) differentiate a tubular body (TB) which stops at the level of the joint membrane (JM). In C a longitudinal section of the sensory neuron (SN) between adjacent antennal glands type A (GhA). In D details of a longitudinal section taken at the ciliary region level. CC, ciliary collar; CR, ciliary rootlets; CU, cuticle; IDS, inner dendritic segment; ODS, outer dendritic segment. Scale bars: A and B ¼ 10 mm; C ¼ 5 mm; D ¼ 2 mm.
exceptional glandular activity. In P. favieri, and probably in all species of Paussini (Nagel, 1979; Di Giulio et al., unpublished data), this is observable from the external by the presence of a great number of pores, different in structure and position, spread throughout the antenna, surrounding the base of single modified setae, crowded in special tegumental areas, grouped at the base of tufts of hairs, or crowded inside deep pockets that store the secretions, with filiform material similar to a paste arising from them. The ultrastructural analysis confirms the complexity of the antennal glandular apparatus of P. favieri, with different types of exocrine glands localized in different regions of the antenna, or mixed without a clear connection to a specific area. Glands type A (GhA) are widespread under the antennal cuticle, mainly concentrated on the ventral side and on symphilous organs. Glands Type B (GhB) deeper within the antennal lumen, particularly in the region close to the antennal pockets. Glands Type C (GhC) have been found deep in the antennal lumen, close to the area housing trophocytes. The general structure of the different
gland types refers to Class 3 secretory cells, according to the classification of insect epidermal glands proposed by Noirot and Quennedey (1974) and Quennedey (1998). In fact, in all cases (GhC are located) each glandular unit is composed of one (GhA) or two (GhB) secretory cells and a duct cell. The secretion produced by the secretory cells is then carried by a cuticular evacuating duct and released through external pores. The occurrence of antennal glands has been reported in several families of coleopterans (Yung, 1938; Matthes, 1970; Cammaerts, 1974; Martin, 1975). Furthermore, the studies carried out using TEM techniques have revealed that antennal glands in Coleoptera belong either to Class 1 (Skilbeck and Anderson, 1994) or Class 3 (Bartlet et al., 1994; Giglio et al., 2005). However, in P. favieri there is an exceptional development of antennal glandular structures in terms of glandular types and their abundance. Despite the large number of secretory cells, we did not find any structure related to the storage of the secretion. However, we can hypothesize that the large receiving canal of GhA could serve as temporary storage for the secretion. The ultrastructural
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features of the secretory cells, i.e. the presence of rough endoplasmic reticulum associated with Golgi apparatus, and the abundance of electron-dense vesicles (at least in GhB) strongly suggest a proteinaceous nature for the secretion. The absence of secretory products within the cytoplasm of GhA secretory cells could be interpreted as a discontinuous activity as regards the secretion, and this was found for the secretory cells of GhB as well. The abundance of the secretion on the antennal surface in all the investigated specimens strongly supports a low or absent volatility for the secretory products. Moreover, the presence of trophocytes with inclusions of large fat vesicles could be interpreted as a storage to supply the energy for the high metabolic activities of the gland cells. It is worth to note that the modified setae present on antennal surface and mostly those that compose the tufts of the symphilous organs are functional mechanoreceptors. Actually, most previous authors working on Paussini and other myrmecophiles used the term ‘‘trichomes’’ for such structures (see Geiselhardt et al., 2007 and references therein). Though not always explicitly defined, this term would refer to uninnervated hairs only functioning in dispensing chemical secretions from underlying glands. Since no muscles have been found associated with the different glands, a feedback control system for the release of the secretions, involving also the mechanosensory hairs, can be hypothesized. The substances produced by the antennal glands of Paussini do not exclusively consist of fat and volatile chemicals (e.g. ethereal oils), like for example in some myrmecophilous Staphilinidae, but represent solid fine ‘‘foods’’ of unknown chemical composition (Nagel, 1979). These exudates are often spread by modified setae, singly or grouped into tufts actively liked by the ants in a ‘‘friendly’’ way (Nagel, 1979; Geiselhardt et al., 2007). Similar tufts of setae and exocrine glands are also present in other parts of the body like frons, prothorax, elytra and pygidium. The substances are thought to contain attractive allomones probably important to avoid aggressions and to induce the host to accept them inside the nests (Geiselhardt et al., 2007). Thus, the specific function (appeasment, adoption, chemical mimicry, food source, etc.) remains speculative or anecdotical (see Geiselhardt et al., 2007, for a review). The complexity and the variety of the antennal glands of P. favieri lead us to the hypothesis that more than one substance can be produced by different areas of the same antenna (also testified by the different electron densities of the secretory product). It is also possible that different glandular complexes can be active in different moments of the symbiotic interaction. Though no significant differences have been observed between males and females in the number and structure of antennal glands, a function for the intersexual communication cannot be excluded, besides the acknowledged myrmecophilous function. Acknowledgments We are grateful to Federica Turco (Queensland Museum, Brisbane, Australia) for her precious help in field collection and rearing of the specimens of Paussus favieri studied in this work. We also thank Dorotea De Propris (Rome, Italy) for her assistance in SEM preparation and observations, and the discussions on paussine antennal microstructures. TEM data were obtained at the CUME, Perugia University (Italy), SEM data at LIME of ‘‘Roma Tre’’ University (Rome, Italy). We thank Augusto Vigna Taglianti (University ‘‘La Sapienza’’ Rome, Italy) and Achille Casale (University of Sassari, Sassari, Italy) for their permission to publish the drawing of P. favieri (Fig. 1) and Wendy Moore (University of Arizona, Tucson, USA) for the linguistic revision of the manuscript.
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