Fine Structure and Chemical Analysis of the Metathoracic Scent Glands of Eurygaster Maura (Linnaeus, 1758) (Heteroptera: Scutelleridae)
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Folia biologica (Kraków), vol. 55 (2007), No 3-4 Fine Structure and Chemical Analysis of the Metathoracic Scent Gland of Eurygaster maura (Linnaeus, 1758) (Heteroptera: Scutelleridae) Dilek DURAK and Yusuf KALENDER Accepted April 16, 2007 DURAK D., KALENDER Y. 2007. Fine structure and chemical analysis of the metathoracic scent glands of Eurygaster maura (Linnaeus, 1758) (Heteroptera: Scutelleridae). Folia biol. (Kraków) 55: 133-141. The morphology and ultrastructure of the metathoracic scent glands (MTG) of Eurygaster maura were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Also, extracts of the volatile fraction of the MTG secretion from males and females were analyzed by capillary gas chromatography-mass spectrometry (GC-MS). In SEM investigations, MTG are composed of a reservoir and a pair of lateral glands connected to the reservoir by a duct. MTG are open in between the meso- and the metacoxae. These areas, called evaporation areas, are composed of mushroom-like elements. In TEM investigations, the reservoir walls contained two types of cells. Generally, a reservoir is lined by a single layer of epithelial cells, type I cells, which have numerous organelles. Type II cells are found only in a certain area of the reservoir wall. These cells have large secretory ducts lined by a cuticular intima layer. The lateral glands are lined by secretory cells and a secretory duct found in their cytoplasm. Nuclei of secretory cells are closed to the basal region of the cells and circular-shaped. In GC-MS investigations, the MTG exhibited a typical scutellerid composition. In general, (E)-2-hexanal, (E)-2-hexenyl acetate, n-tridecane, n-hexanoic acid, octadecanoic acid, and n-dodecane compounds were present, while diisooctyl acetate and 14-Beta-H-Pregna were detected only in the male extracts of Eurygaster maura. Key words: Heteroptera, Eurygaster maura, scent glands, TEM, gas chromatography-mass spectrometry. Dilek DURAK, Bozok University, Faculty of Arts and Science, Department of Biology, 06100 Divanliyolu, Yozgat, Turkey. E-mail: [email protected] Yusuf KALENDER, Gazi University, Faculty of Arts and Science, Department of Biology, 06500 Teknikokullar, Ankara, Turkey. E-mail: [email protected] Stink bugs, as their name suggests, are character- the stink bugs because they usually retaliate by dis- ized by the production of large quantities of charging volatile secretions from their MTG when strong-smelling and irritating defensive chemicals, they are disturbed or molested. In general, chemi- which are released when the bugs are disturbed or cal analyses were done on most of the stink bugs. molested (ALDRICH 1988). Odorous compounds But there are few studies on the ultrastructure of are produced by both adults and immature stages the scent glands. (STADDON 1979; PAVIS et al. 1994; HO et al. In this paper, the metathoracic scent glands 2001) and numerous reports attest to their efficacy (MTG) were studied by scanning and transmission as defense against predation (KRALL et al. 1999; electron microscopy and the volatiles were ana- HO et al. 2001). They also may be used as alarm lyzed by GC-MS. pheromones (KOU et al. 1989), demonstrated for similar types of compounds produced by bug spe- cies in other families (GUNAWARDENA & Material and Methods BANDUMATHIE 1993; LEAL et al. 1994). A single or a pair of median or ventral scent Insect material glands is usually found in the metathoracic region of Hemiptera (CARAYON 1971). Eurygaster maura Adult Eurygaster maura were collected from is a cosmopolitan species and lives on the various Gramineae in Ayas, Ankara, Turkey, dur- Gramineae (LODOS 1986). These bugs are impor- ing June through September, 2005. Insects were tant pests of economic crops. They are known as reared and maintained at 22-24 oC and 70% r.h. 134 D. DURAK,Y.KALENDER o with a 12:12 light-dark photoperiodic regime in hydride (CaH2) and stored at -20 C. Extracts were plastic jars in the laboratory. Bugs were main- analyzed (~2 Fl of the extract) by splitless coupled tained on fresh host-plants until dissection. The in- gas chromatography-mass spectrometry (GC-MS) sects were dissected in insect saline (0.7% NaCl + with a Agilent 6890 series fitted with a HP-5 MS 0.3% KCl) (SANTOS-MALLET & DE SOUZA 1990). column (30m H 0.25 mm I.D. H 0.25 µm film) and interfaced to an Agilent 5973 mass selective detec- tor (electron impact ionization, 106 eV). The GC Scanning electron microscopy (SEM) was programmed at 50 oC/ 2 min then 5 oC/ min to 250 oC, with injector and transfer line tempera- For scanning electron microscopy the thoracic tures of 250 and 280 oC, respectively, with helium region was dissected, the tergites removed and the carrier gas. Compounds were tentatively identi- MTG (reservoir and glands) were fixed for 3 h fied by GC-MS, and identifications were con- with 3% glutaraldehyde in 0.1 M sodium phos- firmed by comparison of the retention times and phate buffer (pH 7.2). After washing in the same mass spectra with those of authentic samples. The buffer, the MTG were post fixed with 1% osmium relative amount of each compound was deter- tetroxide in 0.1 M sodium phosphate buffer, dehy- mined from the area under GC peaks. drated in graded ethanol, dried using 1,1,1,3,3,3- hexamethyldisilazane (HMDS), and coated with gold. The observations were made in a Jeol JSM 5600 scanning electron microscope. Results Transmission electron microscopy (TEM) Scanning electron microscope (SEM) results For transmission electron microscopy the MTG In SEM investigations the MTG of E. maura (reservoir and glands) were dissected and fixed for posses a well developed reservoir and paired 1 h with 3 % glutaraldehyde in 0.1 M sodium phos- glands located in the upper-lateral region of the phate buffer (pH 7.2) at 4 oC. MTG were washed reservoir (Fig. 1). The reservoir is bag-shaped. with sodium phosphate buffer pH 7.2 for 2 h at 4 oC There are irregular projections and intrusions on and postfixed in 1% OsO4 in sodium phosphate its surface. The reservoir is connected to the lateral buffer pH 7.2 for 1 h at 4 oC. Tissue samples were o glands by a canal in the apical surface (Fig. 1). washed with the same buffer for3hat4 C, dehy- nd rd drated in a graded ethanol series and embedded in The ostioles located between the 2 and 3 coxae Araldite. Thin sections were cut with a Leica EM open to the outside. The MTG open through paired UC6 (Leica Co., Austria) ultramicrotome. The ostioles. In E. maura ostioles have a globular sections were viewed and photographed on a Jeol shape. A groove-like structure extends down- 100 CX II transmission electron microscope (Jeol wards from the ostiole (Fig. 2). While this struc- Ltd, Japan) at 80 kV. ture is long and wide, its ostiole is circular. This structure is named the ostiolar groove or peri- treme. After secretion through the ostiole, the con- Chemical analysis tents of the MTG spread out from this region via the ostiolar groove. Therefore the ostiole, the os- The MTG were removed and immersed in ~100 tiolar groove and their surroundings are termed the Fl analytical grade hexane distilled from calcium evaporation area. Mushroom-like structures exist Fig. 1. Scanning electron micrograph of metathoracic scent glands of E. maura. LG – lateral gland, R – reservoir. Bar=500 Fm. Metathoracic Scent Gland of Eurygaster maura 135 Fig. 2. Ostiole (arrow), ostiolar duct (OD) and evaporation areas (Ý)inE. maura. Bar=500 Fm. Fig. 3. Evaporation areas in E. maura. Bar=10 Fm. Fig. 4. Single layer of cylindirical epithelial cells (Type I cells) in the reservoir of E. maura. M – mitochondria, N – nucleus, V – vacuole, In – intima, _ – mineral concretion, H 12500. 136 D. DURAK,Y.KALENDER Fig. 5. Type II cell and seceretory ducts (SD) in the reservoir of E. maura. M – mitochondria, H 8750. Fig. 6. General appearance of secretory cells of lateral gland of E. maura. L – lumen, M – mitochondria, N – nucleus, Sg – secretion granule, Av – autophagic vacuole, H 8750. Fig. 7. Secretory ducts of secretory cell of lateral gland of E. maura. N – nucleus, Sg – seceretion granule, Sc – secretory canalicule, H 7500. Metathoracic Scent Gland of Eurygaster maura 137 on the surface of the evaporation area (Fig. 3). and located in the apical-basal direction. It is as- These globular structures are slightly concave in sumed that these ducts transport the secretion of the center and have irregular projections. The the epithelial cells to the reservoir (Fig. 5). mushroom-like structures are linked to each other Lateral glands examined by TEM show that the by ridges and numerous trabecules found under the cells are arranged around a lumen which is star- ridges (Fig. 3). like in shape and narrow (Fig. 6). It is lined by an epicuticular intima layer. Three to five cells are Transmission electron microscope (TEM) results found around a single lumen. Cells are surrounded by a common basal lamina. These are the secretory The reservoir of MTG is lined by a single layer of cells and their nuclei are located close to the basal columnar epithelial cells. These cells are called region of the cells and circular-shaped. The secre- Type I cells (Fig. 4) and are surrounded by a thin tory material was of different size and the number basal lamina and a cuticular intima layer on the of mitochondria in the cytoplasm was variable apical surface. The cuticular intima layer does not (Fig. 6). The secretory cells have an intracellular have a flattened structure, it possesses projections canalicule around a central lumen in their cyto- and intrusions. Nuclei of reservoir cells are gener- plasm (Fig.