Comparative fine structural analysis of the male reproductive accessory glands in Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae)

Abstract

The morphology and ultrastructure of the male reproductive accessory glands from Bactrocera oleae and Ceratitis capitata were comparatively investigated. In both insects, there are two types of glands, mesoderm‐ and ectoderm‐derived, which open in the ejaculatory duct. The mesoderm‐derived glands are sac‐like in B. oleae and very long tubules in C. capitata, whereas the ectodermic glands, generally branched finger‐like structures, are longer in B. oleae than in C. capitata. Despite their different morphology, the ultrastructure of the two types of glands is quite similar in both Tephritids. The epithelium of the mesoderm‐derived glands consists of binucleate and microvillate secretory cells. In C. capitata, but not in B. oleae, the secretory cells contain smooth endoplasmic reticulum and, in the sexually mature males, enlarged polymorphic mitochondria. The gland lumen is filled with a dense or sometimes granulated secretion. The ectoderm‐derived glands undergo a cycle of maturation, by which their epithelial cells form a large subcuticular cavity filled with an electron‐transparent secretion. Electrophoretic analysis of accessory gland secretion reveals in both species the presence of low molecular weight protein bands. A major band of about 29 kDa or 30 kDa in B. oleae and C. capitata, respectively, is revealed.

Keywords:

• Olive fly
• medfly
• insect reproduction
• seminal fluid
• accessory gland secretion
• male reproductive system

Introduction

The olive fly Bactrocera oleae (Rossi) (Diptera, Tephritidae) is the most serious insect pest of olive fruit in the world. Primarily known in the Mediterranean area, recently it has been found in California and Mexico (Rice 2000; Phillips & Rice 2001; Mavragani‐Tsipidou 2002). Damage to the olive fruit is caused by larvae which, growing (and sometime pupating) in the fruit, destroy the mesocarp and cause the fruit dropping, thus affecting the quality and the quantity of the oil or the production of table olives (Koveos & Tzanakakis 1990; Rice 2000).

The conventional method to control the olive fly using chemical pesticides, although effective, has heavy reliance on the environment and possible implication for human welfare and health (Denholm & Rowland 1992). Thus, during the last few decades, there has been great interest in developing environment‐friendly methods and strategies to control this economically important insect, such as the use of natural insecticides, Bacillus thuringiensis (Alberola et al. 1999), entomophagous insects (Calvitti et al. 2002), mass trapping systems (Haniotakis 1986; Ferrari et al. 2003) and the Sterile Insect Technique (SIT; Kapatos 1989). However, the SIT, already developed and applied to control the medfly C. capitata, appears difficult to transfer to B. oleae because of the lack of an effective and low‐cost mass‐rearing method for this species (Tzanakakis 1989). The reproductive biology of this species, the knowledge of which is of basal importance in developing low environmental impact, species‐specific methods of control (Gilmore 1989; Robinson 2002), is not yet well understood. Only some studies deal with the reproductive biology of Bactrocera genus (Tzanakakis et al. 1968; Mazomenos 1989; Zouros & Loukas 1989; Kuba & Ito 1993) and few data are available on the ultrastructure of the reproductive apparatus of both male (De Marzo et al. 1976) and female (Solinas & Nuzzaci 1984; Dallai et al. 1993; Nezis et al. 2001) B. oleae.

Recently, we described the ultrastructure of the male reproductive accessory glands from C. capitata and reported a preliminary characterization of their secretions (Marchini et al. 2003). The male accessory gland secretions, at least in Diptera, including B. oleae and C. capitata, transmitted to the female during copulation, are responsible for two main behavioural and physiological effects: repression of sexual receptivity to further mating and egg laying stimulation (Cavalloro & Delrio 1970; Delrio & Cavalloro 1979; Chen 1984; Jang 1995; Wolfner 1997; Miyatake et al. 1999).

In this paper we describe the morphology and the ultrastructure of the male reproductive accessory glands in B. oleae in comparison to those in the related Tephritid species C. capitata. A preliminary investigation on the protein components of the gland secretion from the olive fly is also reported.

Materials and methods

Insects

Pupae of B. oleae were field collected in South Tuscany and Apulea (Italy). Adults were maintained in laboratory conditions, at 20 ± 1 °C, 60–70% relative humidity with a photoperiod cycle of 12 h light: 12 h dark, fed with the liquid medium used for C. capitata (see below). C. capitata flies were reared at 25 ± 1 °C in 60–70% relative humidity with a photoperiod cycle of 14 h light: 10 h dark. Larvae were grown on a carrot powder–brewer's yeast‐based diet and adults were fed with a liquid medium essentially consisting of sugar and amino acids (Marchini 1990, Ph.D. Thesis, Siena University).

Light microscopy

The morphology of the male reproductive accessory glands from B. oleae and C. capitata of different ages (newly emerged or 10–20 days old) was investigated in both fresh and fixed samples by using a Leica DMRB light microscope equipped with Nomarski optics. Males were dissected in 0.1 M Na‐phosphate buffer (PB) at pH 7.2 or in phosphate‐buffered saline. Fixation was carried out in 2.5% glutaraldehyde in PB overnight at 4 °C. Fresh samples were mounted on glass slides in PB; fixed samples in 50% glycerol in PB.

Oil red O staining was done on whole‐mount samples. Accessory glands from 15–20‐day‐old males from B. oleae or C. capitata were fixed in 2.5% glutaraldehyde in PB plus 3% sucrose for 1.5 h at 4°C. After rinsing in PB, samples were transferred to distilled water and equilibrated in 60% isopropanol at room temperature for 5 min before staining in 0.3% Oil red O in 60% isopropanol at room temperature for 25 min. Accessory glands were then washed with 60% isopropanol and rinsed with distilled water. Mounting was done as described above.

Alcian Blue staining was also performed on whole‐mount samples from flies of the same age as above. Accessory glands, fixed in 4% formaldehyde in PB overnight at 4 °C and rinsed in PB and distilled water, were incubated in the staining solution (1% Alcian Blue in 3% acetic acid, pH 2.5) for 25 min at room temperature. After rinsing samples in distilled water, mounting was carried out as described above.

Scanning (SEM) and transmission (TEM) electron microscopy

For SEM preparations, the reproductive accessory glands from males of both insect species (newly emerged or 10–15 days old) were fixed in 2.5% glutaraldehyde plus 1.8% sucrose in PB at 4°C overnight, washed in the same buffer and post‐fixed in 1% OsO₄ in PB for 1 h at 4 °C. Samples were then dehydrated in a graded ethanol series and dried by the critical point method in a Balzers CPD 010 apparatus. Material was mounted, gold‐coated in a Balzers MED 010 sputtering unit and observed with a Philips XL20 at 10 kV.

For TEM observations, after dissecting, fixing and dehydrating samples as described above, material was transferred in propylene oxide as bridging solvent and embedded in epon‐araldite. A better preservation of the epithelium from the mesodermic accessory glands was obtained by using 2.5% glutaraldehyde in PB plus 3% sucrose. Ultrathin sections, performed by a Reichert Ultracut II E ultramicrotome, were stained with uranyl acetate and Pb‐citrate. Observations were performed with a Philips CM10 TEM at 80 kV.

Recovery of secretion from accessory glands and electrophoresis

Sexually mature (10–15 days old) males from B. oleae and C. capitata were dissected in PB at pH 6.8 to isolate the reproductive apparatus. Accessory glands were further dissected and processed as previously described (Marchini et al. 2003) to recover their secretion. Sample protein concentration was determined according to Bradford (1976).

SDS–polyacrylamide gel electrophoresis was carried out on 20% polyacrylamide gels according to Laemmli (1970). Before running, samples (5 male equivalent, mesodermic plus ectodermic gland secretions) were denatured in dissociation buffer (Laemmli 1970), boiled for 5 min and spun at 12,000 g for 2 min at room temperature to remove eventual debris. Low molecular weight standards ranging from 2.5 to 31 kDa or from 2.5 to 45 kDa were purchased from Promega and Amersham Pharmacia Biotech, respectively. Proteins were revealed by Sypro Ruby protein gel stain (Molecular Probes).

Results

Gross morphology

In both B. oleae and C. capitata, the male reproductive system consists of pear‐like testes, long and narrow deferent ducts and accessory glands which flow into a common large chamber; from this chamber starts a long ejaculatory duct which ends in the erecting and pumping organ (Hanna 1938; figure 1A–B). The accessory glands have a different shape in the two species and consist of two types: ectoderm‐ and mesoderm‐derived glands.

Figure 1 SEM micrographs of male reproductive system fromBactrocera oleae (A) and Ceratitis capitata (B). Two types of glands are visible: the ectodermal accessory glands (eag) and the mesodermal accessory glands (mag), respectively. Abbreviations: ej, ejaculatory duct; eo, erecting and pumping organ; d, deferent duct; T, testis.

In B. oleae the ectodermic glands, with a spongy appearance and their distal part bent (figure 1A; see also figure 6A, C), are distributed as one dorsal and two ventral pairs. They measure about 50 μm in diameter and 0.3–1.5 mm in length. The glands of the dorsal pair are long (1–1.5 mm) and generally branched at different levels along their length. On the ventral side, a pair of glands, 0.3–0.6 mm in length, usually branched, can be sometimes asymmetrically substituted on one side by a single 50 μm long lobation. The third pair of ectodermic glands is constituted of single or distally branched units, of variable length but generally longer with respect to the glands of the previous pair. The mesodermic glands are two sac‐like structures 600–800 μm long and 80 μm in diameter (figure 1A). They enter the common chamber of the ejaculatory duct between the dorsal and the ventral pairs of ectodermic glands.

In C. capitata the ectodermic glands consist of some short finger‐like and a pair of claviform spongy structures which are 150–300 μm long and 50–65 μm wide (finger‐like) or about 70 μm long and 30 μm wide (claviform; figure 1B). The finger‐like glands, often branched at their insertion in the common chamber, can vary in number and are not paired: generally, on the dorsal side a two‐branched gland is found, whereas on the ventral side a two‐branched and a single gland are present. The mesoderm‐derived glands are constituted of a pair of slender, long tubular structures (18 mm long and 30 μm wide) which distally adhere to the Malpighian tubules by a net of tracheoles (figure 1B).

Ultrastructure

The fine structure of the two types of glands is consistently different.

In both species, epithelial cells of all the ectoderm‐derived glands (Figures 2A–D, 3A,B, 5A,B) undergo a maturation cycle by reaching sexual maturity.

Figure 2 TEM micrographs of male reproductive ectodermal accessory glands fromBactrocera oleae. A,B, newly emerged male; C,D, 15‐day‐old male. A, Cross‐section through epithelial cells, lined by a thin cuticle (ct) beneath which the apical plasma membrane forms deep infoldings delimiting narrow cavities. These cavities are lined by long microvilli (mv). A large nucleus (N) is visible in the basal region where the plasma membrane gives origin to short invaginations. A thin basal lamina and muscle fibres (Ms) are visible beneath the epithelial cells. G, Golgi system. B, Detail of the apical region of the secretory cells. Note the layer of loose material beneath the epicuticle (ct) where microvilli (mv) end. The epicuticle is interrupted by pores (arrowheads). The limiting circumvoluted plasma membranes are associated with mitochondria (m). At the bottom of the figure, many microvilli from the apical infoldings are visible. C, Cross‐section through secretory cells showing a large cavity (cv) delimited by long and thin microvilli (mv). The lateral cytoplasm of neighbouring cells is reduced to a thin layer separated by a fine basal lamina (arrows). Basally, the cell contains mitochondria (m) and vesicles. The apical epicuticle is also visible (arrowhead). D, Detail of the apical region of the secretory cell showing the perforated epicuticle (arrowheads) and the apex of the laminar cytoplasm of two adjacent cells. Note the circumvoluted limiting plasma membrane, the mitochondria (m) and the microvilli (mv) with different orientation.

Figure 3 TEM micrographs of reproductive accessory glands fromCeratitis capitata sexually mature male. A,B, ectodermal glands; C,D, mesodermal glands. A, Cross‐section through a secretory cell. The cytoplasm is reduced to a thin layer surrounding a large central cavity (cv) where the secretion is stored. Microvilli (mv) line the cavity and expand up to the cuticle (ct). Beneath the cells a muscle layer (Ms) is visible. N, nucleus. B, Detail of the apical region of secretory cells. The adjacent cells are joined by an apical zonula adherens (arrowhead) and a long septate junction (sj). In the cytoplasm, mitochondria (m) and rough endoplasmic reticulum (rer) are visible. mv, long and thin microvilli. C, Cross‐section through epithelial cells surrounding a narrow lumen (L). In the cytoplasm numerous polymorphic large mitochondria (m), often close to the limiting plasma membrane, are recognizable; clusters of smooth endoplasmic reticulum (ser) and large vesicles of electron‐transparent material are visible. N, nucleus. D, Detail of the secretory cell showing the labyrinthine system of the intercellular spaces (asterisks). In the region, a large mitochondrion provided with paracrystalline material in its matrix (arrow) and a large amount of parallel‐aligned cysternae of smooth endoplasmic reticulum (ser) are visible. N, nucleus; m, mitochondria.

In newly emerged flies of B. oleae, the epithelial cells of the ectodermic glands show deep infoldings in the apical plasma membrane to form a few small cavities filled with slender microvilli (Figures 2A,B, 5A). The cells, provided with a large spheroidal nucleus, are joined together by sinuous limiting membranes along which septate junctions are visible. The cytoplasm is rich in mitochondria, endoplasmic reticulum and Golgi systems (figure 2A). The cells are lined by a cuticular intima consisting of an outer thin dense epicuticular layer and a loosely fibrous layer beneath, variable in thickness from 40 to 200 nm (figure 2B). The outer layer is interrupted by pores; at their level the epicuticular layer infolds to form an opening about 85 nm wide (figure 2B). In newly emerged males from C. capitata, the general organization of secretory cells (not shown) is very similar to that of B. oleae (figure 5A). However, the fibrous layer beneath the thin outer epicuticle is not recognizable so that microvilli can reach the most apical cuticular layer. The epithelial cells lay on a thin basal lamina and are surrounded by muscle fibres (Figures 2A, 3A).

In aged males (about 15–20 days old) from both species the epithelial cells change dramatically in shape with the appearance of a characteristic large subcuticular cavity which reduces the cytoplasm to slender layers on lateral and basal regions (Figures 2C, 3A,B, 5B). In such a cavity an electron transparent secretion is stored (Figures 2C, 3A). Microvilli are still evident as long thin projections towards the inner part of the cavity. The basal region of the cell contains a flattened nucleus and packed mitochondria (Figures 2C, 3A). In B. oleae, in the apical region, the pores present in the thin epicuticle are smaller than in the earlier stage (50 nm vs 85 nm) and the fibrous cuticular layer seems to be less evident so that microvilli can reach the outer epicuticular layer (figure 2D).

The mesoderm‐derived glands, in both species, are provided with binucleated and microvillated epithelial cells (figure 4A). The fine structural organization, however, is markedly different in the two Tephritids.

Figure 4 TEM micrographs of male mesodermal accessory glands fromBactrocera oleae. A, newly emerged male. B,C, 15‐day‐old male. A, Cross‐section through a binucleated (N), microvillated (mv) secretory cell showing several mitochondria in the cytoplasm. The limiting plasma membranes show long junctional contacts (arrows) while in the basal region the intercellular space expands (asterisk). Several vesicles are visible in the luminal cytoplasm. The epithelial cells lie on a basal lamina and externally exhibit muscle fibres (Ms). L, gland lumen. B, Cross‐section through a secretory cell showing a cytoplasm rich in mitochondria (m), rough endoplasmic reticulum (rer), Golgi systems (G), and a large secretory mass (Se) near the apex. Ms, muscle fibres. C, Apical region of a secretory cell at the time of extrusion of a secretory mass (Se). G, Golgi system; rer, rough endoplasmic reticulum; m, mitochondria.

Figure 5 Schematic drawing of secretory cells from ectodermic accessory glands in bothB. oleae and C. capitata. A, newly emerged male. B, sexually mature male. Arrowheads indicate the pores in the apical cuticle. N, nucleus; cv, cavity; mv, microvilli; m, mitochondria; rer, rough endoplasmic reticulum.

Figure 6 Whole mounts(dorsal view) of male ectodermic (eag) and mesodermic (mag) accessory glands from B. oleae (A,C) and C. capitata (B,D) stained with Alcian Blue (A,B) or Oil red (C,D). Acidic mucopolysaccharides are detectable in both ectodermic and mesodermic glands from C. capitata, but only in the ectodermic glands from B. oleae. Lipids are exclusively revealed in the lumen of the mesodermic gland from C. capitata.

In B. oleae, by reaching sexual maturity, the cytoplasm enriches in rough endoplasmic reticulum, Golgi complexes and mitochondria (compare figure 4A with figure 4B). Moreover, in the newly emerged flies small vesicles of secretion are evident in the apical region of the secretory cells (figure 4A), discharged in the lumen by a macroapocrine mechanism. In the aged males, the secretory activity greatly increases and large masses of secretions are formed and stored in the luminal cytoplasm; these masses are further extruded in the lumen (figure 4B,C).

In C. capitata, on the contrary, the cytoplasm is less abundant in rough endoplasmic reticulum and, instead, it is comprised clusters of smooth endoplasmic reticulum (figure 3C,D), Golgi systems and large mitochondria which contains in their matrix paracrystalline inclusions (figure 3C,D). The secretion from the apical cell surface is represented by small vesicles and large portions of cytoplasm protruding into the lumen (not shown). In the gland lumen of B. oleae, a homogeneous, fine granular electron‐transparent material is stored (figure 4A–C), while in C. capitata the gland lumen contains a dense material (figure 3C), sometimes with a granular appearance.

Histochemistry and electrophoresis

To detect the presence of acidic mucopolysaccharides in the secretion of the accessory glands from the two Tephritids, Alcian Blue staining was performed on both types of glands (figure 6A,B). Staining was positive only for the ectodermic glands from B. oleae (figure 6A), whereas the two types of glands were intensively stained in C. capitata (figure 6B).

Lipids, revealed by the Oil red O staining (figure 6C,D), were detected only in the mesodermic glands from C. capitata (Marchini et al. 2003).

A denaturing SDS–PAGE analysis of the male accessory gland secretion from B. oleae and C. capitata was performed to compare their protein profile. We recovered about 0.8 μg total protein/male in the secretion from ectodermic plus mesodermic accessory glands of both B. oleae and C. capitata. We used a 20% polyacrylamide gel to look for protein bands with a molecular weight compatible with that of the sex peptide from Drosophila (about 3 kDa). The outcome of the experiment is shown in figure 7: the protein pattern evidenced polypeptides ranging in molecular weight from 7.5 or 10 kDa to over 45 kDa in B. oleae and C. capitata, respectively. Molecular weight of protein bands over 31 kDa was determined using markers up to 45 kDa (not shown). We focused our attention on protein bands of molecular weight  30 kDa which, at least in C. capitata, are most likely transferred to the female during copulation (Marchini et al. 2003). A major band with molecular weight of 29 and 30 kDa, respectively, was detectable in both B. oleae and C. capitata. A 25 kDa protein band was more intensively stained in B. oleae than in C. capitata. In B. oleae, moreover, two other bands were evident, with molecular weight of 17.5 and 7.5 kDa, respectively. In C. capitata, on the contrary, in addition to a minor band of about 10 kDa, also present in B. oleae, a 16 kDa protein band was shown, whereas no lower molecular weight protein was visible.

Figure 7 Protein profile of male reproductive accessory gland secretion fromCeratitis capitata (Cc ag) and Bactrocera oleae (Bo ag) after SDS–PAGE. Each sample corresponds to accessory gland secretion (mesodermic plus ectodermic glands) from five males (about 4 μg total protein). Low molecular weight markers (MW), 1 μg/each protein. Numbers indicate molecular weights, expressed in kilodaltons. Arrowheads indicate the major band of 30 kDa or 29 kDa in C. capitata and B. oleae, respectively.

Discussion

The male accessory gland system of B. oleae and C. capitata consists of two types of structures: ectodermic and mesodermic glands. The former, with different size and shape in the two species, are, however, recognizable for their spongy appearance at first glance and their ultrastructural organization. Ectodermic glands were previously described as a group of three pairs in some Tephritid species (Hanna 1938; Drew 1969; De Marzo et al. 1976; Delrio & Cavalloro 1979). However, in C. capitata only the claviform glands are indeed a pair; the other ectodermic glands, either branched or single, finger‐like units, consist of two asymmetric groups of glands, dorsally or ventrally inserted into the common chamber of the ejaculatory duct. Nevertheless, also in B. oleae where the presence of three pairs of glands has been confirmed, sometimes the symmetry is lost by the middle pair of ectodermic glands in which a two‐branched gland can be substituted on one side by a single lobation.

The mesodermic glands, organized as one pair, are very long and tubular‐shaped in C. capitata and sac‐like in B. oleae. These last glands correspond to the “sacciformi” glands by De Marzo et al. (1976).

In both species, the epithelial cells of the ectodermic glands are characterized by a marked change in their ultrastructure in newly emerged compared to sexually mature flies. In fact cells, which have cytoplasm rich in endoplasmic reticulum, mitochondria and Golgi systems in newly emerged males, progressively reduce their cytoplasm to give origin to a subcuticular cavity bordered by thin microvilli. At sexual maturity, these cells consist of a large cavity surrounded by a thin layer of cytoplasm. The cuticle lining the cells apically is perforated by minute pores through which a secretion flows out. Then, the contraction of the muscle fibres surrounding the glands would allow the secretions to flow into the ejaculatory duct. The presence of the cuticle on the epithelial cells indicates the ectodermic origin of these glands. Such cuticle was not well described by previous authors, who interpreted it as a granular secretion (De Marzo et al. 1976). In the sexually mature male of both species this cuticle appears quite comparable. However, in the newly emerged males, before the retraction of the cytoplasm to form the subcuticular cavity, beneath the thin epicuticle a fibrous layer is present in B. oleae but not in C. capitata. The ultrastructural organization of these glands, which have only one type of cell producing a secretion and discharging it into the gland lumen, resembles that of ectodermal glands of type I described by Noirot & Quennedey (1974). The ectodermic glands seem to be active in proteinaceous secretion according to their richness in cytoplasmic organules involved in such type of secretions. The presence of acidic mucopolysaccharides in the ectodermic glands of B. oleae, hypothesized by De Marzo et al. (1976), has been confirmed by the Alcian Blue staining. Such staining is instead negative in the mesodermic glands. On the contrary, acidic mucopolysaccharides are present in both ectodermic and mesodermic glands from C. capitata in which the presence of polysaccharides were already evidenced in the two type of glands by Thiery's reaction (Marchini et al. 2003).

Mesodermic glands, despite having binucleated epithelial cells, are quite different in their ultrastructure in the two examined species. In C. capitata the epithelial cells have abundant smooth endoplasmic reticulum and are consequently involved in the production of a secretion containing lipids (Marchini et al. 2003). In B. oleae, on the contrary, they exhibit a cytoplasm very rich in rough endoplasmic reticulum while the smooth endoplasmic reticulum has not been revealed. The ultrastructural data are supported by Oil red staining for detection of neutral lipids which revealed a negative reaction in both type of glands from B. oleae, unlike in C. capitata where secretion inside mesodermic glands appears strongly positive (Marchini et al. 2003). In both species, however, the secretion is stored in the gland lumen after flowing through the apical cytoplasm by a macroapocrine mode of secretion.

The functional activity of the secretions produced by the male accessory glands is a matter of great interest and discussion. It is now clear that in several insects, mainly in Diptera, male accessory gland secretions, which contribute to seminal fluid and are transferred to the female by copulation, play a role in the induction of behavioural and physiological modification in females. Mated females are not able to remate for a certain period and are stimulated to lay a higher number of eggs compared to virgin females (Leopold 1976; Chen 1984; Kubli 1996; Bloch Qazi et al. 2003). In Drosophila, a particular type of accessory gland proteins (Wolfner 1997; Wolfner et al. 1997), named sex peptide (Chen et al. 1988; Kubli 1992), is responsible for these effects. In B. oleae, in the melon fly B. cucurbitae and in C. capitata, a substance in seminal fluid was strongly suggested to be responsible for suppression of female receptivity to a second mating (Cavalloro & Delrio 1970; Delrio & Cavalloro 1979; Kuba & Ito 1993). In C. capitata, accessory gland secretion was demonstrated to be responsible for such physiological effect (Jang 1995; Miyatake et al. 1999).

A preliminary biochemical investigation by electrophoresis on the accessory gland secretion from B. oleae and C. capitata was carried out in the attempt to detect some sex peptide‐like molecules. The electrophoresis analysis performed on the total gland secretion from the two Tephritids does not reveal strong differences in protein profile showing several bands, most of which ranging from 46 to 7.5 kDa. A major band with a similar molecular weight (29 and 30 kDa, respectively) is detectable in both B. oleae and C. capitata. Such protein band, at least in C. capitata, seems to be related to the mesodermic glands (Marchini et al. 2003). Although low molecular mass proteins are present in both species, and a very low molecular weight band (7.5 kDa) is present in B. oleae, peptides with a mass compatible with that of the sex peptide from Drosophila (about 3 kDa; Kubli 1992) were not detected. However, peptides of molecular weight lower than 10 kDa for C. capitata or 7.5 kDa for B. oleae could be present in gland secretion, but in amount too low to be revealed by electrophoresis stainings. Moreover, in other insects, molecules with function of sex peptide having a molecular weight higher (about 8 kDa) than in Drosophila have been identified (Smid et al. 1997; Lee & Klowden 1999).

In a previous paper (Marchini et al. 2003) we showed that in C. capitata, as well as in Drosophila (Bairati 1968; Lung & Wolfner 2001), transfer of the accessory gland secretion to the female begins before that of sperm: a conspicuous amount of secretion is transferred after 30 min copulation (mean time of copulation is ∼ 3 h), whereas at the same time no sperm are found in the fertilization chamber and only a very small amount of sperm has reached the spermathecal receptacles (Marchini et al. 2001). In addition to an immediate passage of sex peptide‐like substances to the female, transferring the sperm fluid before the spermatozoa could have the function to lubricate the female storage organs favouring optimal conditions to receive the spermatozoa. Indeed, the presence in B. oleae and C. capitata of acidic mucopolysaccharides, in association or not with lipids, could have such a role other than facilitating sperm movement into the female reproductive tract.

We are now studying the male accessory gland secretion in C. capitata and B. oleae with the aim of identifying some protein components with a sex peptide function.

Acknowledgements

We wish to thank R. Dallai (University of Siena, Italy) for precious advice and critical reading of the manuscript. We also thank Professor G. Nuzzaci (University of Bari, Italy) for providing us with a wild population of B. oleae from Apulea. This research was supported by University of Siena Funds (PAR) to D.M.