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Morphology of the sternal gland in workers of Coptotermes gestroi (Isoptera, Rhinotermitidae)
Micron 37 (2006) 551–556 www.elsevier.com/locate/micron
Morphology of the sternal gland in workers of Coptotermes gestroi (Isoptera, Rhinotermitidae) A.M. Costa-Leonardo Departamento de Biologia, Instituto de Biocieˆncias, UNESP, Av. 24A, No. 1515, Bela Vista, Cep 13506-900, Rio Claro, SP, Brasil Received 26 July 2005; received in revised form 2 December 2005; accepted 3 December 2005
Abstract The sternal gland is considered the only source of trail pheromones in termites. The morphology of the sternal gland was investigated in workers of Coptotermes gestroi using transmission and scanning electron microscopy. The results showed a small bilobed gland at the anterior part of the fifth abdominal sternite. The cuticular surface of the sternal gland showed a V-shaped structure with two peg sensilla in elevated socket and various campaniform sensilla. Pores and cuticular scale-like protuberances also occur in the glandular area. The ultrastructure showed a gland composed of class 1 cells and two different types of class 3 cells distinguished by location, different size and electron-density of secretory vesicles. Small class 3 cells (type 1) of the anterior lobe are inserted among class 1 cells and have weakly electron-dense vesicles associated with mitochondria, glycogen and smooth endoplasmic reticulum. The class 3 cells (type 2) of posterior lobe showed many round electron-lucent vesicles of secretion, abundant free ribosomes and a well-developed Golgi apparatus. Each class 3 cell is connected to the cuticle by a cuticular duct constituted by the receiving canal and the conducting canal. The secretion of class 1 cells is stored in an inner subcuticular reservoir that is delimited by the microvilli of these cells. This inner reservoir is large and crossed by the campaniform sensilla and ducts of two types of class 3 cells that open outside of the insect body. An exterior reservoir also is present between the fourth and fifth sternite. The complex structure of the sternal gland suggests multicomponents for the trail pheromone in the worker of C. gestroi. # 2005 Elsevier Ltd. All rights reserved. Keywords: Termite; Rhinotermitidae; Trail pheromone; Exocrine gland
1. Introduction Termites are eusocial insects and the maintenance of this sociality depends mainly on their chemical communication. The pheromones involved in this chemical communication are little known in Isoptera. In addition, few morphological studies are available regarding to the exocrine glands that are the source of pheromones. The termite sternal gland was first described by Grassi and Sandias (1893 apud Noirot, 1969) and occurs in all castes and developmental stages, although not functional in some of them (Oloo, 1981; Ignatti and Costa-Leonardo, 2001; Sˇobotnı´k and Hubert, 2003). This gland is unique and appears in the histological sections as an epidermal thickening of the sternite. The ultrastructure of the sternal gland is available for few Isoptera species (Quennedey, 1971a,b; Quennedey and Leuthold, 1978) and some descriptions of this gland morphology
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have been misinterpreted in the past (Quennedey, personal information). A single sternal gland is located under the fifth sternite in the Kalotermitidae, Rhinotermitidae, Serritermitidae and Termitidae, while in the Hodotermitidae and Termopsidae, it is located under the fourth sternite. The termites of the Mastotermitidae family present three glands situated under the third, fourth and fifth sternites (Noirot, 1995). To date, the sternal gland of the workers has been described as the only source of trail pheromones (Stuart, 1963; Runcie, 1987), however, this gland is also involved in sexual communication of the alates (Noirot, 1969). Rosengaus et al. (2004) suggest that the secretions of sternal gland had the original function of controlling microorganisms in the termite nest, whilst its role in communication evolved secondarily. The termite trails are used in the recruitment of the individuals towards the food and water sources, areas of disturbance and repairing or building of galleries and nests (Kaib, 1999; Kaib et al., 1982). Few compounds have been identified as trail pheromones of Isoptera, although there is
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some evidence that termite trail communication is based on a pheromonal blend (Runcie, 1987; Kaib et al., 1982). Ho¨lldobler and Carlin (1987) suggest an anonymous signal as major component and other species-specific compounds as being constituents of sternal gland secretion. Dodecatrienol has been identified as the major component of the trail-following pheromone in some Rhinotermitidae (Tokoro et al., 1989; Laduguie et al., 1994; Wobst et al., 1999; Bordereau et al., 1993) and neocembrene A has been reported to be the main component in the genus Prorhinotermes (Sillam-Dusse`s et al., 2005) and of some Termitidae (Moore, 1974; Birch et al., 1972; McDowell and Oloo, 1984). The trailfollowing pheromone of fungus-growing termites has been identified to contain dodecenol and dodecadienol (Peppuy et al., 2001a,b). However, species-specific compounds have not been isolated or identified until now in termites (Peppuy et al., 2001a). This morphological investigation attempts to further the general understanding of termite exocrine glands, as well to disclose the basic ultrastructure of the sternal gland in workers of Coptotermes gestroi. 2. Material and methods Workers of C. gestroi (Wasmann, 1896) were collected with cardboard traps at Rio Claro (228230 S, 478320 W), SP, Brazil. For scanning electron microcopy, worker abdomens were fixed in FAA fluid (formol, alcohol and acetic acid; 1:3:1), dehydrated in 50, 70, 95 and 100% acetone baths and criticalpoint dried. The samples were then coated with gold and observed under a Philips scanning electron microscope. For transmission electron microscopy, a piece of the worker abdomen, containing the gland, was fixed in 2% glutaraldehyde and 4% paraformaldehehyde in 0.2 M sodium cocadilate buffer and postfixed in 1% osmium tetroxide preceded by dehydration in a graded acetone series. The samples were previously stained with uranyl acetate, embedded in epon-araldite and sectioned with a Reichert Leica ultramicrotome. Double-stained thin sections were observed in a CM 100 Philips electron microscope. Semi-thin sections, stained with 1% methylene blue and azur II were used for light microscopy.
Fig. 1. Longitudinal semi-thin section of the sternal gland showing the anterior (al) and posterior (pl) lobes. ga, nervous ganglion; *, exterior reservoir; IV, V, sternites.
The sternal gland of C. gestroi appears as a thickening of the epidermis at the anterior part of the fifth sternite and its globous anterior lobe is covered by the long fourth sternite (Figs. 1, 3 and 4). Between the fourth and fifth sternite there is a space named exterior reservoir (Fig. 1) because it is located outside the insect body. The ultrastructure analyses showed a gland composed of classes 1 and 3 cells (Fig. 3) according to the classification of Noirot and Quennedey (1974, 1991). The anterior lobe is less developed and constituted by colunar cells (Fig. 3). The class 1 glandular cells present round nuclei and compacted microvilli in the apical region. Smooth endoplasmic reticulum and mitochondria with characteristic cristae are abundant in these cells. The contact between class 1 cells is formed by an apical
3. Results The workers of C. gestroi present a small bilobed sternal gland located in front of the fourth abdominal ganglion (Fig. 1). The results of scanning microscopy reveal the glandular area as a V-shaped structure in the anterior part of the fifth sternite. A detailed view of the glandular surface shows two peg sensilla (Fig. 2) in a elevated socket, one on each side of the gland. In addition, campaniform sensilla appear in the lateral parts of the gland. Pores also occur in the glandular area (Fig. 2) and most of them are located in depressions near the campaniform sensilla. Cuticular scales like protuberances are predominant in the anterior region of the gland, although they also occur in the posterior part.
Fig. 2. Detail of the glandular region surface in the fifth sternite of Coptotermes gestroi worker. Arrows, pores; ca, campaniform sensilla; cs, cuticular scales; p, peg sensilla.
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Fig. 3. Schematic organization of the sternal gland. ec, epithelial cell; er, exterior reservoir; ir, inner reservoir; cs, campaniform sensillum; C1, class 1 cell; C3, class 3 cell of anterior lobe; C’3, class 3 cell of posterior lobe; IV, fourth sternite; V, fifth sternite.
zonula adherens followed by septate junction. Class 3 cells (type I) are inserted among class 1 cells in the anterior lobe of the gland. These class 3 cells are small and their cytoplasm is full of secretory vesicles weakly electron-dense associated with mitochondria (Figs. 3 and 5). Smooth endoplasmic reticulum is also observed among the secretion. Often, glycogen is found around the secretory vesicles (Fig. 6). Deep invaginations of plasmic membrane are present in the basal region of these cells. Class 3 glandular cells, different to those described in the anterior lobe, are widespread in the posterior lobe of the sternal gland (Fig. 3). These cells present many round electron-lucent vesicles of secretion (Figs. 7–10), abundant free ribosomes and a well-developed Golgi apparatus (Fig. 10). Each class 3 cell is connected to the cuticle by a duct that has two parts, one corresponding to the receiving canal (endapparatus) and the other to the conducting canal. The membrane of these cells present microvilli arranged around the receiving canal (Figs. 8 and 9), that is formed by a layered
Fig. 4. Sagital section of the sternal gland with the large subcuticular space (*) full of secretion (s). The cuticle (c) of this region is modified and very thick. Cells of class 3 (arrows) predominate in the posterior lobe of the sternal gland. IV, V, sternites.
Figs. 5 and 6. (5) Small class 3 cells (arrows) with the secretory vesicles (s) and mitochondria (m). cc, conducting canal. (6) Detail of the secretory vesicles (s) in small class 3 cells. Note the glycogen (g) disposed around the secretion.
porous cuticle (Fig. 9). On the other hand, the conducting canals are surrounded by canal cells and show an electron-dense and continuous cuticular layer (Fig. 11). These canals cross the cuticle and open outside of the insect body, permitting the discharging of the secretion to the exterior reservoir. The receiving canal is short in the anterior class 3 cells (type 1) but it is sinuous in the posterior class 3 cells (type 2) and, may appear more than once in a plane section (Fig. 8). The fourth abdominal ganglion is located very close to the sternal gland (Figs. 1 and 7). Further, a group of sensorial cells are located at the anterior lobe of the sternal gland and correspond to the cells of campaniform sensilla (Fig. 3). A large subcuticular space full of secretion also appears in the anterior lobe, close to the median part of the sternal gland and was called inner reservoir (Figs. 3 and 4). This reservoir is delimited by the microvilli of class 1 glandular cells and is crossed by the ducts of class 3 glandular cells and campaniform sensilla. The cuticle of this region is elevated and strongly electron-dense, and it is the opening of the ducts from different class 3 glandular cells. The inner reservoir is separated from the exterior reservoir by this modified cuticle.
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Figs. 7 and 8. (7) Posterior lobe of the sternal gland with another type of class 3 cells. Note the cytoplasm full of round electron-lucent secretion (s). ga, nervous ganglion; n, nucleus. (8) Nucleus (n), secretory vesicles (s) and receiving canal (rc) of the posterior class 3 cells. mv, microvilli.
Small epidermal cells are arranged close to the cuticle in the posterior lobe of the sternal gland (Fig. 12). These cells are slightly flattened and form a continuous layer crossed by canals placed between glandular epithelium and the cuticle. Their cytoplasm contains free ribosomes and small mitochondria. 4. Discussion The sternal gland of C. gestroi is relatively complex, despite its small size. In all studied species of Rhinotermitidae the gland is bilobed and its development depends on the species as well as the caste (Smythe and Coppel, 1966; Noirot, 1969; Grasse´, 1982; Ampion and Quennedey, 1981). However, the sternal gland is not functional in larval instars and undergoes degeneration in physogastric queens (Grasse´, 1982; Ignatti and Costa-Leonardo, 2001). The clear organization of this gland, mainly the differences among secretory cells, is not observed with light microscopy (Smythe and Coppel, 1966), but size alterations may be estimated (Oloo, 1981; Costa-Leonardo and Kitayama, 1990). Exocuticular structures on the sternal gland, similar to those observed in the present study, were also described by Liang
Figs. 9 and 10. (9) Cross-section of a receiving canal (rc) from posterior class 3 cell. mv, microvilli; s, secretory vesicle. (10) Detail of the Golgi (G) and secretory vesicles (s) from posterior class 3 cell.
et al. (1979) for Coptotermes formosanus and some species of Reticulitermes. The majority of the pores are found in depressions close to the campaniform sensilla that are situated in a specific glandular region. The pores are connected to class 3 secretory cells and the arrangement of these structures may vary among different species. The campaniform sensilla are mechanoreceptors and may be stimulated by pressure or bending of the insect cuticle. Probably, they play a regulatory role in the trail deposition since provide termite information about the amount of pheromone released during trail laying. According to Liang et al. (1979) the peg sensilla seem to be chemoreceptors and probably also have some function in the trail pheromone deposition. Further ultrastructure study will elucidate this subject. The scale-like protuberances seem not to participate in the deposition of the trail pheromone, since the previous sternite overlaps the gland surface entirely during trail laying (Quennedey, 1975). Three types of glandular cells have been described in insects (Noirot and Quennedey, 1974; Quennedey, 1998). Class 1 cells are modified epidermal cells and their secretion must pass through the outer cuticle to be released outside the insect body. The secretion produced by class 2 glandular cells is transferred to one or two epidermal cells before crossing the outer cuticle. Class 3 cells posses a duct lined by cuticle which is continuous with the external surface of the insect
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Figs. 11 and 12. (11) Conducting canals (*) of class 3 cells crossing the cuticle (c). cca, canal cell. (12) Posterior lobe of the gland with the small epidermal cells (ec) disposed under the cuticle (c) and class 3 cells with the electron-lucent secretory vesicles (s). cc, conducting canals.
body. These cells are always associated with duct cells and their secretion is eliminated to the outside through the cuticular ducts. Previous studies by Quennedey (1971a, 1972) on the sternal gland of Kalotermes flavicollis and Trinervitermes geminatus indicated the presence of class 2 cells, which are not common in the insect exocrine glands. Class 2 cells were not observed in the present study of the sternal gland of C. gestroi. According to Noirot and Quennedey (1991), these cells are considered to be modified epidermal oenocytes and are found in some sternal glands of Isoptera. Class 2 secretory cells also occur in tergal and posterior sternal glands of termites and cockroaches (Sreng, 1984, 1985; Bordereau et al., 2002; Sˇobotnı´k and Weyda, 2005). These cells were not found in the genus Mastotermes but in other termites such cells were described spread among the class 1 cells, as in the case of Kalotermes, or forming a central aggregate, as in the case of Hodotermes. In addition, the class 3 cells are scarce or they are absent in the sternal gland of Termitidae.
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Quennedey (1971b), studing the ultrastructure of the worker sternal gland of Reticulitermes santonensis and Schedorhinotermes putorius, found four types of secretory cells, two different cells with ducts that are class 3 cells, cells of class 2 and intercalary cells, that are class 1 cells. This author described the anterior lobe of the gland, consisting of classes 1–3 (type 1) cells, and the posterior lobe of class 3 (type 2) cells. These cells showed distinct granules of secretion and one of the class 3 cells also had glycogen associated with the secretory vesicles. A detailed description of the sternal gland in workers of Psammotermes hybostoma showed classes 1 and 2 cells in the anterior part of the gland and two types of class 3 cells in the posterior part (Quennedey, 1998). As in workers of C. gestroi, secretions of class 1 cells are emitted into a dome-shaped region where a modified cuticle overlies an inner subcuticular reservoir. The secretions of class 1 cells are accumulated in the subcuticular reservoir and, only later, are eliminated through the cuticle. The inner extracellular space located under the cuticle with the function of storing secretion is also present in all the termites of Rhinotermitidae family studied so far (Quennedey, 1971b; Ampion and Quennedey, 1981). Glandular secretory products showed great variability in shape and electron-density, as occur in the secretory vesicles present in the different class 3 cells of the C. gestroi sternal gland. According to Quennedey (1998), the electron-lucent secretion of the termite sternal gland contains lipids and this also seems to be the case in C. gestroi workers, since the lipids are the precursors of many pheromones. Mitochondria may be abundant and can reach a considerable size, forming a significant part of the cellular content in some exocrine glands (Quennedey, 1972, 1998; Gracioli et al., 2004). In various insect glands, the close contact of the mitochondria with the secretion is common and, recently, some authors have described the transformation of mitochondria into secretory granules, as observed in the ducts of labial glands of P. simplex (Sˇobotnı´k and Weyda, 2003). The strategies of foraging are varied in Isoptera and the different glandular composition, due mainly to specific trail pheromones, appears to be responsible for the great structure diversity of the termite sternal gland. The complex morphology of the sternal gland in workers of C. gestroi suggests that the secretion is constituted by different and elaborate components that are responsible for the trail behavior and orientation of the foragers. Acknowledgements This study was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico–CNPq. The author thanks A. Yabuki for technical assistance. References Ampion, M., Quennedey, A., 1981. The abdominal epidermal glands of termites and their phylogenetic significance. In: Howse, P.E., Cle´ment, J.-L. (Eds.), Biosystematics of Social Insects. Academic Press, London, pp. 249–261.
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Birch, A.J., Brown, W.V., Corrie, J.E.T., Moore, B.P., 1972. Neocembrene-A, a termite trail pheromone. J. Chem. Soc. 1, 2653–2658. Bordereau, C., Robert, A., Bonnard, O., Le Que´re´, J.-L., 1993. De´tection du (Z,Z,E)-3,6,8-dodecatrien-1-ol par les ouvriers et les essaimants de deux espe´ces de termites champignonnistes: Pseudacanthotermes spiniger et P. militaris (Isoptera, Macrotermitinae) Actes Coll. Insectes Sociaux 8, 145– 149. Bordereau, C., Cancello, E.M., Se´mon, E., Courrent, A., Quennedey, B., 2002. Sex pheromone identified after solid phase microextraction from tergal glands of female alates in Cornitermes bequaerti (Isoptera, Nasutitermitidae). Insectes Sociaux 49, 209–215. Costa-Leonardo, A.M., Kitayama, K., 1990. Sternal gland size related to polyethism in the termite Constrictotermes cyphergaster (Termitidae, Nasutitermitinae). Cieˆncia e Cultura 42 (11), 975–977. Gracioli, L.F., Silva de Moraes, R.L.M., Cruz-Landim, C., 2004. Ultrastructural aspects of the mandibular gland of Melipona bicolor Lepeletier, 1836 (Hymenoptera: Apidae, Meliponini) in the castes. Micron 35, 331– 336. Grasse´, P.-P., 1982. Termitologia, vol. 1. Masson, Paris, p. 676. Ho¨lldobler, B., Carlin, N., 1987. Anonymity and specificity in the chemical communication signals of social insects. J. Comp. Physiol. 161, 567– 581. Ignatti, A.C., Costa-Leonardo, A.M., 2001. The exocrine glands of swarming females and physogastric queens of Cornitermes cumulans (Kollar) (Isoptera, Termitidae, Nasutitermitinae). Sociobiology 18 (4), 1089–1096. Kaib, M., 1999. Termites. In: Hardie, J., Minks, A.K. (Eds.), Pheromones of Non-lepidopteran Insects Associated with Agricultural Plants. CABI Publishing, Wallingford, pp. 329–353. Kaib, M., Bruinsma, O., Leuthold, R.H., 1982. Trail-following in termites: evidence for a multicomponent system. J. Chem. Ecol. 8 (9), 1193–1205. Laduguie, N., Robert, A., Bonnard, O., Vieau, F., Le Que´re´, J.L., Se´mon, E., Bordereau, C., 1994. Isolation and identification of (3Z, 6Z, 8E)-3,6,8Dodecatrien-1-ol in Reticulitermes santonensis Feytaud (Isoptera, Rhinotermitidae): roles in worker trail-following and in alate sex-attraction behavior. J. Insect Physiol. 40, 781–787. Liang, M.Y., Coppel, H.C., Matsumura, F., Esenther, G., 1979. Exocuticular structures on the sternal gland segments of Rhinotermitidae. Sociobiology 4 (2), 169–189. McDowell, P.G., Oloo, G.W., 1984. Isolation, identification, and biological activity of trail following pheromone of termite Trinervitermes bettonianus (Sjo¨stedt) (Termitidae: Nasutitermitinae). J. Chem. Ecol. 10, 835– 851. Moore, B.P., 1974. Pheromones in termite societies. In: Birch, M.C. (Ed.), Pheromones. American Elsevier, New York, pp. 250–266. Noirot, Ch., 1969. Glands and secretions. In: Krishna, K., Weesner, F. (Eds.), Biology of Termites, vol. 1. Academic Press, New York, pp. 89–123. Noirot, Ch., 1995. The sternal glands of termites: segmental pattern, phylogenetic implications. Insectes Sociaux 42, 321–323. Noirot, Ch., Quennedey, A., 1974. Fine estructure of insect epidermal glands. Ann. Rev. Entomol. 19, 61–80. Noirot, Ch., Quennedey, A., 1991. Glands, gland cells, glandular units: some comments on terminology and classification. Annales de la Socie´te´ Entomologique de France 27 (2), 123–128. Oloo, G.W., 1981. The sternal gland: variation in size and activity in worker instars of Trinervitermes bettonianus (Sjost) (Termitidae). Insect Sci. Appl. 2, 145–147. Peppuy, A., Robert, A., Se´mon, E., Bonnard, O., Truong Son, N., Bordereau, C., 2001a. Species specificity of trail pheromones of fungus-growing termites from northern Vietnam. Insectes Sociaux 48, 245–250. Peppuy, A., Robert, A., Se´mon, E., Ginies, C., Lettere, M., Bonnard, O., Bordereau, C., 2001b. (Z)-Dodec-3-en-1-ol, a novel termite trail pheromone
identified after solid phase microextration from Macrotermes annandalei. J. Insect Physiol. 47, 445–453. Quennedey, A., 1971a. Les glandes exocrines des termites. I. Etude histochimique et ultrastructurale de la glande sternale de Kalotermes flavicollis Fab. (Isoptera, Kalotermitidae) Z. Zellforsch 121, 27–47. Quennedey, A., 1971b. Les glandes exocrines des termites. II. Organisation de la glande sternale des Rhinotermitidae. Etude ultrastructurale pre´liminaire. Compts Rendus de L’Academie de Sciences. Serie D 273, 376– 379. Quennedey, A., 1972. Les glandes exocrines des termites. III. Structure fine de la glande sternale de Trinervitermes geminatus Wasman (Termitidae, Nasutitermitinae) Z. Zellforsch. 130, 205–218. Quennedey, A., 1975. Morphology of exocrine glands producing pheromones and defensive substances in subsocial and social insects. In: Noirot, C., Howse, P.E., Masne, G.Le (Eds.), Pheromones and Defensive Secretions in Social Insectes. Symp., IUSSI, Dijon, pp. 1–21. Quennedey, A., 1998. Insect epidermal gland cells: ultrastructure and morphogenesis. In: Harrison, F.W. (Ed.), Microscopic Anatomy of Invertebrates, vol. 11. Wiley-Liss Inc. Press, New York, pp. 177–207. Quennedey, A., Leuthold, R.H., 1978. Fine structure and pheromonal properties of the polymorphic sternal gland in Trinervitermes bettonianus (Isoptera, Termitidae). Insectes Sociaux 25 (2), 153–162. Rosengaus, R.B., Traniello, J.F.A., Lefebvre, M.L., Maxmen, A.B., 2004. Fungistatic activity of the sternal gland secretion of the dampwood termite Zootermopsis angusticollis. Insectes Sociaux 51, 259–264. Runcie, C., 1987. Behavioral evidence for multicomponent trail pheromone in the termite Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae). J. Chem. Ecol. 13, 1967–1978. Sillam-Dusse`s, D., Se´mon, E., Moreau, C., Valterova´, I., Sˇobotnı´k, J., Robert, A., Bordereau, C., 2005. Neocembrene A, a major component of the trailfollowing pheromone in the genus Prorhinotermes (Insecta, Isoptera, Rhinotermitidae). Chemoecology 15, 1–6. Smythe, R.V., Coppel, H.C., 1966. A preliminary study of the sternal gland of Reticulitermes flavipes (Isoptera: Rhinotermitidae). Ann. Entomol. Soc. Am. 59, 1008–1010. Sˇobotnı´k, J., Hubert, J., 2003. The morphology of the exocrine glands of Prorhinotermes simplex (Isoptera: Rhinotermitidae) and their ontogenetical aspects. Acta Societatis Zoologicae Bohemicae 67, 83–98. Sˇobotnı´k, J., Weyda, F., 2003. Ultrastructural ontogeny of the labial gland apparatus in Prorhinotermes simplex (Isoptera: Rhinotermitidae). Arthropod Struct. Dev. 31, 255–270. Sˇobotnı´k, J., Weyda, F., 2005. Ultrastructural study of tergal and posterior sternal glands in Prorhinotermes simplex (Isoptera: Rhinotermitidae). Eur. J. Entomol. 102, 81–88. Sreng, L., 1984. Morphology of the sternal and tergal glands producing the sexual pheromones and the aphrodisiacs among the cockroaches of the subfamily Oxyhaloinae. J. Morphol. 182, 279–294. Sreng, L., 1985. Ultrastructure of the glands producing sex pheromones of the male Nauphoeta cinerea (Insecta, Dictyoptera). Zoomorphology 105, 133– 142. Stuart, A.M., 1963. Origin of the trail in the termites Nasutitermes corniger (Motschulsky) and Zootermopsis nevadensis (Hagen). Physiol. Zool. 36, 69–84. Tokoro, M., Tsunoda, K., Yamaoka, R., 1989. Isolation and primary structure of trail pheromone of the termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Wood Res. 76, 29–38. Wobst, B., Farine, J.P., Ginies, C., Se´mon, E., Robert, A., Bonnard, O., Conne´table, S., Bordereau, C., 1999. (3Z, 6Z,8E)-3,6,8-Dodecatrien-1-ol, a major component of the trail-following pheromone in two sympatric termite species Reticulitermes lucifugus grassei Cle´ment and R. santonensis Feytaud. J. Chem. Ecol. 25, 1305–1318.
Morphology of the sternal gland in workers of Coptotermes gestroi (Isoptera, Rhinotermitidae) A.M. Costa-Leonardo Departamento de Biologia, Instituto de Biocieˆncias, UNESP, Av. 24A, No. 1515, Bela Vista, Cep 13506-900, Rio Claro, SP, Brasil Received 26 July 2005; received in revised form 2 December 2005; accepted 3 December 2005
Abstract The sternal gland is considered the only source of trail pheromones in termites. The morphology of the sternal gland was investigated in workers of Coptotermes gestroi using transmission and scanning electron microscopy. The results showed a small bilobed gland at the anterior part of the fifth abdominal sternite. The cuticular surface of the sternal gland showed a V-shaped structure with two peg sensilla in elevated socket and various campaniform sensilla. Pores and cuticular scale-like protuberances also occur in the glandular area. The ultrastructure showed a gland composed of class 1 cells and two different types of class 3 cells distinguished by location, different size and electron-density of secretory vesicles. Small class 3 cells (type 1) of the anterior lobe are inserted among class 1 cells and have weakly electron-dense vesicles associated with mitochondria, glycogen and smooth endoplasmic reticulum. The class 3 cells (type 2) of posterior lobe showed many round electron-lucent vesicles of secretion, abundant free ribosomes and a well-developed Golgi apparatus. Each class 3 cell is connected to the cuticle by a cuticular duct constituted by the receiving canal and the conducting canal. The secretion of class 1 cells is stored in an inner subcuticular reservoir that is delimited by the microvilli of these cells. This inner reservoir is large and crossed by the campaniform sensilla and ducts of two types of class 3 cells that open outside of the insect body. An exterior reservoir also is present between the fourth and fifth sternite. The complex structure of the sternal gland suggests multicomponents for the trail pheromone in the worker of C. gestroi. # 2005 Elsevier Ltd. All rights reserved. Keywords: Termite; Rhinotermitidae; Trail pheromone; Exocrine gland
1. Introduction Termites are eusocial insects and the maintenance of this sociality depends mainly on their chemical communication. The pheromones involved in this chemical communication are little known in Isoptera. In addition, few morphological studies are available regarding to the exocrine glands that are the source of pheromones. The termite sternal gland was first described by Grassi and Sandias (1893 apud Noirot, 1969) and occurs in all castes and developmental stages, although not functional in some of them (Oloo, 1981; Ignatti and Costa-Leonardo, 2001; Sˇobotnı´k and Hubert, 2003). This gland is unique and appears in the histological sections as an epidermal thickening of the sternite. The ultrastructure of the sternal gland is available for few Isoptera species (Quennedey, 1971a,b; Quennedey and Leuthold, 1978) and some descriptions of this gland morphology
E-mail address: [email protected]. 0968-4328/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2005.12.006
have been misinterpreted in the past (Quennedey, personal information). A single sternal gland is located under the fifth sternite in the Kalotermitidae, Rhinotermitidae, Serritermitidae and Termitidae, while in the Hodotermitidae and Termopsidae, it is located under the fourth sternite. The termites of the Mastotermitidae family present three glands situated under the third, fourth and fifth sternites (Noirot, 1995). To date, the sternal gland of the workers has been described as the only source of trail pheromones (Stuart, 1963; Runcie, 1987), however, this gland is also involved in sexual communication of the alates (Noirot, 1969). Rosengaus et al. (2004) suggest that the secretions of sternal gland had the original function of controlling microorganisms in the termite nest, whilst its role in communication evolved secondarily. The termite trails are used in the recruitment of the individuals towards the food and water sources, areas of disturbance and repairing or building of galleries and nests (Kaib, 1999; Kaib et al., 1982). Few compounds have been identified as trail pheromones of Isoptera, although there is
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some evidence that termite trail communication is based on a pheromonal blend (Runcie, 1987; Kaib et al., 1982). Ho¨lldobler and Carlin (1987) suggest an anonymous signal as major component and other species-specific compounds as being constituents of sternal gland secretion. Dodecatrienol has been identified as the major component of the trail-following pheromone in some Rhinotermitidae (Tokoro et al., 1989; Laduguie et al., 1994; Wobst et al., 1999; Bordereau et al., 1993) and neocembrene A has been reported to be the main component in the genus Prorhinotermes (Sillam-Dusse`s et al., 2005) and of some Termitidae (Moore, 1974; Birch et al., 1972; McDowell and Oloo, 1984). The trailfollowing pheromone of fungus-growing termites has been identified to contain dodecenol and dodecadienol (Peppuy et al., 2001a,b). However, species-specific compounds have not been isolated or identified until now in termites (Peppuy et al., 2001a). This morphological investigation attempts to further the general understanding of termite exocrine glands, as well to disclose the basic ultrastructure of the sternal gland in workers of Coptotermes gestroi. 2. Material and methods Workers of C. gestroi (Wasmann, 1896) were collected with cardboard traps at Rio Claro (228230 S, 478320 W), SP, Brazil. For scanning electron microcopy, worker abdomens were fixed in FAA fluid (formol, alcohol and acetic acid; 1:3:1), dehydrated in 50, 70, 95 and 100% acetone baths and criticalpoint dried. The samples were then coated with gold and observed under a Philips scanning electron microscope. For transmission electron microscopy, a piece of the worker abdomen, containing the gland, was fixed in 2% glutaraldehyde and 4% paraformaldehehyde in 0.2 M sodium cocadilate buffer and postfixed in 1% osmium tetroxide preceded by dehydration in a graded acetone series. The samples were previously stained with uranyl acetate, embedded in epon-araldite and sectioned with a Reichert Leica ultramicrotome. Double-stained thin sections were observed in a CM 100 Philips electron microscope. Semi-thin sections, stained with 1% methylene blue and azur II were used for light microscopy.
Fig. 1. Longitudinal semi-thin section of the sternal gland showing the anterior (al) and posterior (pl) lobes. ga, nervous ganglion; *, exterior reservoir; IV, V, sternites.
The sternal gland of C. gestroi appears as a thickening of the epidermis at the anterior part of the fifth sternite and its globous anterior lobe is covered by the long fourth sternite (Figs. 1, 3 and 4). Between the fourth and fifth sternite there is a space named exterior reservoir (Fig. 1) because it is located outside the insect body. The ultrastructure analyses showed a gland composed of classes 1 and 3 cells (Fig. 3) according to the classification of Noirot and Quennedey (1974, 1991). The anterior lobe is less developed and constituted by colunar cells (Fig. 3). The class 1 glandular cells present round nuclei and compacted microvilli in the apical region. Smooth endoplasmic reticulum and mitochondria with characteristic cristae are abundant in these cells. The contact between class 1 cells is formed by an apical
3. Results The workers of C. gestroi present a small bilobed sternal gland located in front of the fourth abdominal ganglion (Fig. 1). The results of scanning microscopy reveal the glandular area as a V-shaped structure in the anterior part of the fifth sternite. A detailed view of the glandular surface shows two peg sensilla (Fig. 2) in a elevated socket, one on each side of the gland. In addition, campaniform sensilla appear in the lateral parts of the gland. Pores also occur in the glandular area (Fig. 2) and most of them are located in depressions near the campaniform sensilla. Cuticular scales like protuberances are predominant in the anterior region of the gland, although they also occur in the posterior part.
Fig. 2. Detail of the glandular region surface in the fifth sternite of Coptotermes gestroi worker. Arrows, pores; ca, campaniform sensilla; cs, cuticular scales; p, peg sensilla.
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Fig. 3. Schematic organization of the sternal gland. ec, epithelial cell; er, exterior reservoir; ir, inner reservoir; cs, campaniform sensillum; C1, class 1 cell; C3, class 3 cell of anterior lobe; C’3, class 3 cell of posterior lobe; IV, fourth sternite; V, fifth sternite.
zonula adherens followed by septate junction. Class 3 cells (type I) are inserted among class 1 cells in the anterior lobe of the gland. These class 3 cells are small and their cytoplasm is full of secretory vesicles weakly electron-dense associated with mitochondria (Figs. 3 and 5). Smooth endoplasmic reticulum is also observed among the secretion. Often, glycogen is found around the secretory vesicles (Fig. 6). Deep invaginations of plasmic membrane are present in the basal region of these cells. Class 3 glandular cells, different to those described in the anterior lobe, are widespread in the posterior lobe of the sternal gland (Fig. 3). These cells present many round electron-lucent vesicles of secretion (Figs. 7–10), abundant free ribosomes and a well-developed Golgi apparatus (Fig. 10). Each class 3 cell is connected to the cuticle by a duct that has two parts, one corresponding to the receiving canal (endapparatus) and the other to the conducting canal. The membrane of these cells present microvilli arranged around the receiving canal (Figs. 8 and 9), that is formed by a layered
Fig. 4. Sagital section of the sternal gland with the large subcuticular space (*) full of secretion (s). The cuticle (c) of this region is modified and very thick. Cells of class 3 (arrows) predominate in the posterior lobe of the sternal gland. IV, V, sternites.
Figs. 5 and 6. (5) Small class 3 cells (arrows) with the secretory vesicles (s) and mitochondria (m). cc, conducting canal. (6) Detail of the secretory vesicles (s) in small class 3 cells. Note the glycogen (g) disposed around the secretion.
porous cuticle (Fig. 9). On the other hand, the conducting canals are surrounded by canal cells and show an electron-dense and continuous cuticular layer (Fig. 11). These canals cross the cuticle and open outside of the insect body, permitting the discharging of the secretion to the exterior reservoir. The receiving canal is short in the anterior class 3 cells (type 1) but it is sinuous in the posterior class 3 cells (type 2) and, may appear more than once in a plane section (Fig. 8). The fourth abdominal ganglion is located very close to the sternal gland (Figs. 1 and 7). Further, a group of sensorial cells are located at the anterior lobe of the sternal gland and correspond to the cells of campaniform sensilla (Fig. 3). A large subcuticular space full of secretion also appears in the anterior lobe, close to the median part of the sternal gland and was called inner reservoir (Figs. 3 and 4). This reservoir is delimited by the microvilli of class 1 glandular cells and is crossed by the ducts of class 3 glandular cells and campaniform sensilla. The cuticle of this region is elevated and strongly electron-dense, and it is the opening of the ducts from different class 3 glandular cells. The inner reservoir is separated from the exterior reservoir by this modified cuticle.
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Figs. 7 and 8. (7) Posterior lobe of the sternal gland with another type of class 3 cells. Note the cytoplasm full of round electron-lucent secretion (s). ga, nervous ganglion; n, nucleus. (8) Nucleus (n), secretory vesicles (s) and receiving canal (rc) of the posterior class 3 cells. mv, microvilli.
Small epidermal cells are arranged close to the cuticle in the posterior lobe of the sternal gland (Fig. 12). These cells are slightly flattened and form a continuous layer crossed by canals placed between glandular epithelium and the cuticle. Their cytoplasm contains free ribosomes and small mitochondria. 4. Discussion The sternal gland of C. gestroi is relatively complex, despite its small size. In all studied species of Rhinotermitidae the gland is bilobed and its development depends on the species as well as the caste (Smythe and Coppel, 1966; Noirot, 1969; Grasse´, 1982; Ampion and Quennedey, 1981). However, the sternal gland is not functional in larval instars and undergoes degeneration in physogastric queens (Grasse´, 1982; Ignatti and Costa-Leonardo, 2001). The clear organization of this gland, mainly the differences among secretory cells, is not observed with light microscopy (Smythe and Coppel, 1966), but size alterations may be estimated (Oloo, 1981; Costa-Leonardo and Kitayama, 1990). Exocuticular structures on the sternal gland, similar to those observed in the present study, were also described by Liang
Figs. 9 and 10. (9) Cross-section of a receiving canal (rc) from posterior class 3 cell. mv, microvilli; s, secretory vesicle. (10) Detail of the Golgi (G) and secretory vesicles (s) from posterior class 3 cell.
et al. (1979) for Coptotermes formosanus and some species of Reticulitermes. The majority of the pores are found in depressions close to the campaniform sensilla that are situated in a specific glandular region. The pores are connected to class 3 secretory cells and the arrangement of these structures may vary among different species. The campaniform sensilla are mechanoreceptors and may be stimulated by pressure or bending of the insect cuticle. Probably, they play a regulatory role in the trail deposition since provide termite information about the amount of pheromone released during trail laying. According to Liang et al. (1979) the peg sensilla seem to be chemoreceptors and probably also have some function in the trail pheromone deposition. Further ultrastructure study will elucidate this subject. The scale-like protuberances seem not to participate in the deposition of the trail pheromone, since the previous sternite overlaps the gland surface entirely during trail laying (Quennedey, 1975). Three types of glandular cells have been described in insects (Noirot and Quennedey, 1974; Quennedey, 1998). Class 1 cells are modified epidermal cells and their secretion must pass through the outer cuticle to be released outside the insect body. The secretion produced by class 2 glandular cells is transferred to one or two epidermal cells before crossing the outer cuticle. Class 3 cells posses a duct lined by cuticle which is continuous with the external surface of the insect
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Figs. 11 and 12. (11) Conducting canals (*) of class 3 cells crossing the cuticle (c). cca, canal cell. (12) Posterior lobe of the gland with the small epidermal cells (ec) disposed under the cuticle (c) and class 3 cells with the electron-lucent secretory vesicles (s). cc, conducting canals.
body. These cells are always associated with duct cells and their secretion is eliminated to the outside through the cuticular ducts. Previous studies by Quennedey (1971a, 1972) on the sternal gland of Kalotermes flavicollis and Trinervitermes geminatus indicated the presence of class 2 cells, which are not common in the insect exocrine glands. Class 2 cells were not observed in the present study of the sternal gland of C. gestroi. According to Noirot and Quennedey (1991), these cells are considered to be modified epidermal oenocytes and are found in some sternal glands of Isoptera. Class 2 secretory cells also occur in tergal and posterior sternal glands of termites and cockroaches (Sreng, 1984, 1985; Bordereau et al., 2002; Sˇobotnı´k and Weyda, 2005). These cells were not found in the genus Mastotermes but in other termites such cells were described spread among the class 1 cells, as in the case of Kalotermes, or forming a central aggregate, as in the case of Hodotermes. In addition, the class 3 cells are scarce or they are absent in the sternal gland of Termitidae.
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Quennedey (1971b), studing the ultrastructure of the worker sternal gland of Reticulitermes santonensis and Schedorhinotermes putorius, found four types of secretory cells, two different cells with ducts that are class 3 cells, cells of class 2 and intercalary cells, that are class 1 cells. This author described the anterior lobe of the gland, consisting of classes 1–3 (type 1) cells, and the posterior lobe of class 3 (type 2) cells. These cells showed distinct granules of secretion and one of the class 3 cells also had glycogen associated with the secretory vesicles. A detailed description of the sternal gland in workers of Psammotermes hybostoma showed classes 1 and 2 cells in the anterior part of the gland and two types of class 3 cells in the posterior part (Quennedey, 1998). As in workers of C. gestroi, secretions of class 1 cells are emitted into a dome-shaped region where a modified cuticle overlies an inner subcuticular reservoir. The secretions of class 1 cells are accumulated in the subcuticular reservoir and, only later, are eliminated through the cuticle. The inner extracellular space located under the cuticle with the function of storing secretion is also present in all the termites of Rhinotermitidae family studied so far (Quennedey, 1971b; Ampion and Quennedey, 1981). Glandular secretory products showed great variability in shape and electron-density, as occur in the secretory vesicles present in the different class 3 cells of the C. gestroi sternal gland. According to Quennedey (1998), the electron-lucent secretion of the termite sternal gland contains lipids and this also seems to be the case in C. gestroi workers, since the lipids are the precursors of many pheromones. Mitochondria may be abundant and can reach a considerable size, forming a significant part of the cellular content in some exocrine glands (Quennedey, 1972, 1998; Gracioli et al., 2004). In various insect glands, the close contact of the mitochondria with the secretion is common and, recently, some authors have described the transformation of mitochondria into secretory granules, as observed in the ducts of labial glands of P. simplex (Sˇobotnı´k and Weyda, 2003). The strategies of foraging are varied in Isoptera and the different glandular composition, due mainly to specific trail pheromones, appears to be responsible for the great structure diversity of the termite sternal gland. The complex morphology of the sternal gland in workers of C. gestroi suggests that the secretion is constituted by different and elaborate components that are responsible for the trail behavior and orientation of the foragers. Acknowledgements This study was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico–CNPq. The author thanks A. Yabuki for technical assistance. References Ampion, M., Quennedey, A., 1981. The abdominal epidermal glands of termites and their phylogenetic significance. In: Howse, P.E., Cle´ment, J.-L. (Eds.), Biosystematics of Social Insects. Academic Press, London, pp. 249–261.
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