Arthropod Structure & Development 33 (2004) 399404 www.elsevier.

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Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand ies (Diptera: Psychodidae: Phlebotominae)
Carolina N. Spiegela, Reginaldo P. Brazilb, Maurilio J. Soaresa,*
rio de Biologia Celular de Microrganismos, Depto. Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz/FIOCRUZ, Laborato 21045-900 Rio de Janeiro, RJ, Brazil b rio de Bioqu mica, Fisiologia e Imunologia de Insetos, Depto. Bioqu mica e Biologia Molecular, Instituto Oswaldo Cruz/FIOCRUZ, Laborato 21045-900 Rio de Janeiro, RJ, Brazil Received 14 January 2004; accepted 23 February 2004
a

Abstract The sex pheromone glands of Lutzomyia cruzi male sand ies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modied into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium potassium iodide (Os KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) proles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand y pheromone biosynthesis. q 2004 Elsevier Ltd. All rights reserved.
Keywords: Lutzomyia; Sand ies; Sex pheromone glands; Ultrastructure

1. Introduction Lutzomyia cruzi (Mangabeira, 1938) sand ies (Diptera: Psychodidae: Phlebotominae) have been incriminated as vectors of visceral leishmaniasis in Mato Grosso do Sul state, Brazil (Santos et al., 1998). L. cruzi is closely related to the L. longipalpis (Lutz and Neiva, 1912) species complex, both belonging to the same subgenus Lutzomyia and to the species series longipalpis (Martins et al., 1978; Young and Duncan, 1994). Females of L. cruzi and L. longipalpis are morphologically indistinguishable and in both species the males present pale spots on their abdominal tergites with sex pheromone disseminating structures similar to small papules, when observed by scanning electron microscopy (Ward et al., 1993; Spiegel et al., 2002). L. cruzi males produce a sex pheromone that has been revealed to be 9-methygermacrene-B (Brazil and Hamilton, 2002), which is also a sex pheromone of one
* Corresponding author. Tel./fax: 55-21-2260-4434. E-mail address: maurilio@ioc.ocruz.br (M.J. Soares). 1467-8039/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2004.02.002

population of the L. longipalpis species complex (Hamilton et al., 1996b). Transmission electron microscopy showed that in L. cruzi the pheromone glands are grouped in the fourth abdominal tergite (Spiegel et al., 2002), as also described in L. longipalpis by Lane and Bernardes (1990). The pheromone gland cells present typical secretory features, such as a large number of mitochondria and the formation of microvilli at the apical plasma membrane. In this work we further analyze the ne structure of the sex pheromone gland cells of L. cruzi males by using cytochemical techniques for the identication of peroxisomes, endoplasmic reticulum (ER) and lipid droplets.

2. Materials and methods 2.1. Insects Lutzomyia cruzi (Mangabeira, 1938) specimens were

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(Mato Grosso do Sul State, Brazil, collected in Corumba 178250 S 488300 W) and were maintained in a colony in our laboratory at 25 ^ 1 8C and 80 ^ 10% r.h. (Brazil et al., 1997). Adult males used in the experiments were 2 to 3 days old. 2.2. Scanning electron microscopy (SEM) L. cruzi adult males from the colonies were sedated for 5 minutes at 2 10 8C and then xed by immersion for at least 2 hours in 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer, pH 7.2. After xation, the insects were washed in 0.1 M cacodylate buffer, pH 7.2, air dried and mounted with adhesive tape to SEM stubs. The specimens were observed in a Zeiss DSM-640 scanning electron microscope. 2.3. Cytochemical detection of lipids Fourth abdominal tergites were dissected and xed for 2 h with 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer, pH 7.2, then rinsed rst in 0.1 M cacodylate buffer and next in 0.1 M imidazole buffer, pH 7.5. The segments were then post-xed for 30 min with 2% osmium tetroxide ller and Fahimi, in 0.1 M imidazole buffer, pH 7.5 (Angermu 1982), rinsed twice in this buffer, dehydrated in graded acetone series and embedded in PolyBed 812 resin. Ultrathin sections were stained for 2 min with lead citrate and then observed by transmission electron microscopy. Tergites routinely xed with 1% osmium tetroxide/0.1 M cacodylate buffer were used as control. 2.4. Cytochemical detection of endoplasmic reticulum Fourth abdominal tergites were dissected and xed for 2 h with 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer pH 7.2, washed once in the same buffer, twice in a solution of 1% potassium iodide (KI) in tridistilled water and then left for 48 h at room temperature in the dark in a 1% OsO4 1% KI solution (Locke and Huie, 1983). Thereafter, the segments were washed with 1% KI in tridistilled water, dehydrated in a graded acetone series and embedded in PolyBed 812 resin. Ultrathin sections were stained for 2 3 min with lead citrate and then observed by transmission electron microscopy. Tergites routinely xed with 1% osmium tetroxide/0.1 M cacodylate buffer were used as control. 2.5. Cytochemical detection of peroxisomes The peroxisomal enzyme marker catalase was detected by incubating the segments in a medium containing diaminobenzidine (DAB), according to Fok and Allen (1975). Fourth abdominal tergites were dissected, xed for 2 h with 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer pH 7.2, washed in 0.1 M cacodylate buffer and in 0.05 M Tris HCl buffer, pH 10.0. Thereafter, they were

incubated for 1 h at room temperature in the dark in 0.05 M Tris HCl buffer, pH 10.0 containing 2 mg/ml DAB, and then for 4 h in this medium containing 0.1% H2O2. At 1 h intervals, the incubation medium was replaced for a new one, containing freshly added H2O2. After incubation, the segments were washed in 0.05 M Tris HCl buffer, pH 10.0, post-xed for 1 h in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.2, washed in the same buffer, dehydrated in a graded acetone series and embedded in PolyBed 812 resin. Control was performed by adding 10 mM aminotrizol, a catalase inhibitor, to the incubation solution. Ultrathin sections were stained for 2 3 min with lead citrate and then observed by transmission electron microscopy.

3. Results Scanning electron microscopy (SEM) showed that sex pheromone disseminating structures are present in the fourth abdominal tergites of L. cruzi adult males, dispersed amidst the microtrichia, appearing as small round cuticular elevations with a central pore (Fig. 1). Observation of L. cruzi adult male tergites processed for the cytochemical detection of lipids by transmission electron microscopy revealed that the apical plasma membrane of the gland cells is invaginated and contains a large number of microvilli, forming a small secretory reservoir. This reservoir is connected to the exterior (the small cuticular papule observed by SEM) by a short cuticular duct. There was a small positive reaction for lipids in between these microvilli (Fig. 2). The central portion of the gland cell cytoplasm presents a large number of electron-lucent vacuoles, not limited by a unit membrane. In routine preparations such vacuoles

Fig. 1. Scanning electron microscopy of tergite IV of L. cruzi. Detail showing the openings (arrow) of the pheromonedisseminating gland structures. Bar 5 mm.

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There are several peroxisomes distributed throughout the gland cell cytoplasm (Figs. 4 and 5), and they are frequently found grouped in large number around the lipid droplets (Fig. 5). Close association between peroxisomes, mitochondria and lipid droplets could be also frequently observed (Fig. 6). Prolonged post-xation with osmium tetroxide (48 h) in the presence of potassium iodide resulted in an effective electron-dense staining of specic cell compartments: the nuclear envelope of the small epithelial cells (Fig. 7) and the gland cells (Figs. 8 and 9), the sarcoplasmic reticulum of the muscle bers (Fig. 8), as well as several ER proles in the secretory cells (Figs. 7 9). There was also a positive reaction inside the microvilli (Fig. 7) and around the peroxisomes (Fig. 9). No reaction was observed in lipid droplets and in the plasma membrane. Background staining was observed as nely granular deposits scattered throughout the cytoplasm.

4. Discussion The sex pheromone of L. cruzi male sand ies is released to the environment from papular cuticular structures present on the fourth abdominal tergites. These structures appear under different morphological aspects in different species of phlebotomines (Ward et al., 1993; Spiegel et al., 2002). The secretion is produced by pheromone gland cells grouped below the cuticle, that were formerly classied as class 3 epidermal gland units (Lane and Bernardes, 1990; Spiegel et al., 2002), according to the nomenclature proposed in the past by Noirot and Quennedey (1974, 1991). However, it has been recently shown that glandular openings can be observed in the cuticle overlaying some class 1 cells (reviewed in Quennedey, 1998, 2000; Quennedey et al., 2002). Thus, in the light of more recent morphological data, it appears that the sex pheromone gland cell of Lutzomyia is a class 1 gland cell with an invagination of apical microvilli. This invagination forms a round reservoir structure, which is connected to the outer environment through a single modication of the thin epicuticle, in the form of a cuticular duct. It is known that lipids represent a rich source of precursors for the pheromone biosynthesis in insects (Percy-Cunningham and MacDonald, 1987; Hoskove et al., 2002). Lipid droplets could be detected in large number in the pheromone cells of L. cruzi, and have been already described in other insect pheromone glands (Percy, 1979; nagy et al., 2001; Ma and Roelofs, Nardi et al., 1996; Fo 2002). It has been shown that changes, both in size and number, of cytoplasmic lipid droplets in the pheromoneproducing cells of the silkmoth Bombyx mori are likely to be in close relationship to pheromone production: there was a daily uctuation in size and number of lipid droplets during the photophase and also depending on age (Fonagy et al., 2001). Our morphological data indicate that pheromone

Figs. 2 and 3. Transmission electron microscopy of L. cruzi pheromone gland cellsCytochemical detection of lipids. Fig. 2: Positive reaction is found in between the microvilli ( ) at the apical cell membrane. Note the cuticular duct (arrow). The asterisks indicate the cytoplasm of epithelial cells. Bar 1 mm. Fig. 3: The cytoplasmic vacuoles present an electron dense positive reaction, demonstrating that they are lipid droplets (L). Note the different densities of some lipid inclusions and the several peroxisomes (P) around them. Bar 1 mm.

present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide resulted in positive (electron dense) reaction for lipids in most gland cell vacuoles (Fig. 3). Occasionally, they were not totally stained, or the electron-density of the vacuoles was variable. The gland cell cytoplasm contains a large number of mitochondria and small round organelles (0.2 0.4 mm in diameter) with a granular, electron-dense matrix (Fig. 3). Positive reaction after incubation in a medium specic for the cytochemical detection of catalase demonstrated that the small electron dense vesicles are peroxisomes (Figs. 4 6).

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components of L. cruzi are synthesized by modications of lipids as precursors, as also described in other insects. This explains the presence of several peroxisomes in the gland cell cytoplasm: these organelles are responsible for different pathways in lipid metabolism and are considered to be involved in the biosynthesis of sex pheromones (Hallberg and Subchev, 1997). The close association between peroxisomes and lipid droplets observed in the sand y pheromone gland cell is not unusual. It is typically found in germinating oil seeds, reecting the main role of peroxisomes on fat mobilization during seed germination (Worgin et al., 1970; De Duve, 1983). The positive reaction with osmium-imidazole in between the microvilli suggests that some components of the gland secretion might be lipids, which is in accordance to chemical analysis of L. longipalpis sex pheromones in male sand ies from Central America, where one of the pheromone components was identied as a fatty acid (Hamilton et al., 1996a). Ultrastrucutural studies of sex pheromone glands of various species of Lepidoptera have established that the presence of smooth ER is a common feature of the modied glandular cells (Percy-Cunningham and Mac Donald, 1987). The nding of an extensive matrix of smooth ER tubules in the sand y gland cell reinforces the role of lipids in the pheromone synthesis in this insect, as the ER is a hallmark of lipid- and steroidsynthesizing cells. Occasionally the tubular arms of the smooth ER cisternae appeared to be distended to form peroxisomes, which is in accordance to the theory on the essential role of the ER in the peroxisomes biogenesis (Titorenko and Rachubinski, 1998). The microvilli of L. cruzi pheromone gland cells appeared electron-dense after prolonged post-xation with osmium tetroxide/potassium iodide, suggesting the presence of inner ER proles. According to Quennedey (1998), two categories of microvilli are distinguishable by their internal organization: in the most common, longitudinal microlaments lie in cytoplasm full of background material. In the other type, the central region is occupied by microlaments around an axial channel continuous with the ER. Thus, our data suggest that the microvilli of L. cruzi pheromone gland cells belong to this second type. In summary, our data indicate that ER, lipid droplets and peroxisomes might be involved in the sand y sex pheromone biosynthesis. Further studies on the development of the pheromone gland cell should be performed to better understand the role of lipids in the pheromone biosynthesis, and the morphogenesis of these gland cells.

Figs. 46. Transmission electron microscopy of L. cruzi pheromone gland cellscytochemical detection of peroxisomes. Fig. 4: General view of the apical portion of the gland cell. Peroxisomes (P) present a positive (electron dense) reaction for catalase. L, lipid droplets. Bar 1 mm. Fig. 5: Note the

large number of peroxisomes distributed in the cell basal portion, around the lipid droplets (L). A muscle cell is observed at the lower part of the micrograph. Bar 1 mm. Fig. 6: Detail of the peroxisomes (P) and mitochondria (M). L, lipid droplet; N, nucleus. Bar 0.5 mm.

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Figs. 79. Transmission electron microscopy of L. cruzi pheromone gland cellscytochemical detection of endoplasmic reticulum. Figs. 7 and 8: The OsKI technique allowed a better visualization of several endoplasmic reticulum proles, as expected in secretory cells. Positive reaction was also found in the envelope of the nuclei (N), in the microvilli ( ), and in the muscle cell ( p ). Fig. 7: Bar 1 mm; Fig. 8: Bar 0.5 mm. Fig. 9: Note the close association between endoplasmic reticulum proles and peroxisomes (P). Bar 0.5 mm.

Acknowledgements The authors would like to thank Dr Beatriz G. Brazil for the constant supply of insects reared in the laboratory. This work was supported by Conselho Nacional de Desenvolvico e Tecnolo gico (CNPq), PAPES-III/FIOmento Cient CRUZ and FIOCRUZ.

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