Morphological and ultrastructural organization of the spermatheca of some Tettigoniidae (Insecta, Orthoptera)

Abstract

A morphological and ultrastructural study of the spermatheca of some species of Orthoptera Tettigoniidae was carried out to understand the role of this female organ in the reproductive biology because no literature exists about it in this insect group. In all the examined species, the spermatheca is of ectodermal origin and is composed of a seminal receptacle, mainly involved in the collection and storage of the spermatozoa, and of a spermathecal duct. In both these organs, the epithelium of the wall is made up of two different cell types: cuticle-forming cells, underlying the cuticular intima, and gland cells. Both of these cell types have secretory features that differ among the species and also within the same species, in relation to the tracts examined. In particular, the ultrastructure of the distal tract of the spermathecal duct indicates a more marked secretory activity than in the other tracts of the duct. This activity is often accompanied by ultrastructural aspects, suggesting a lysis activity in both the epithelium and the upper cuticle. Based on these findings, it is hypothesized that the seminal receptacle and the spermathecal duct have different functional roles, despite having a similar general structural organization.

Keywords:

• Cuticle
• seminal receptacle
• spermiolysis
• spermathecal duct

Introduction

In addition to the gonads, the insect reproductive system includes a considerable number of various secondary structures that are essential for reproduction; in fact, these structures take part in the transfer and protection of gametes. One of the main roles is performed by the spermatheca, which is the organ of the female reproductive system responsible for sperm collection during copulation, sperm storage and their release for fertilization. Furthermore, the spermatheca has also been recognized as the place where morphological and biochemical changes of the male gamete occur, making it ready for fertilization (Longo et al. 1993).

The insect spermatheca is a complex anatomic system of ectodermal origin that shows notable differences in number, shape and structure among the various species. In general, however, it is composed of a seminal receptacle, whose main function is to collect and store sperm, and a spermathecal duct, of various lengths, opening into a bursa copulatrix or a recess in the vagina (Snodgrass 1937; Smith 1968; Chapman 1969; Grassè 1977).

Even though numerous observations have been made on the morphology and structure of the insect spermatheca, data concerning Othoptera Ensifera are referred only for few species (Melis & Dallai 1963, 1966); in addition, no studies exist on the ultrastructural organization of the Tettigoniidae spermatheca. In order to extend our knowledge of the insect spermathecal systems, investigations were made on the morphology and ultrastructure of the spermatheca in some species of Orthoptera Tettigoniidae, to provide a better understanding of its structure and role in the reproductive biology of this insect group.

Materials and methods

Surveys were carried out on the spermathecas of sexually mature females of four species of Orthoptera Tettigonioidea: Platycleis intermedia (Serville), Platycleis grisea (Fabricius), Tessellana tessellata (Charpentier), and Sepiana sepium (Yersin), belonging to the Tettigoniidae family and collected in various locations in eastern Sicily, Italy.

Observations were carried out on the various tracts of the genital spermatheca of each insect.

For in toto observations concerning the morphology of the spematheca, insects were dissected in Ringer solution and the spermathecas were observed using a stereomicroscope; for histological investigations, the spermathecas were then fixed in Carnoy's or in Bouin's liquid, dehydrated in an alcohol series and then included in Histowax. The 5–7 µm sections were stained using Haematoxylin–Eosin and Heidenhain's Azan. No significant differences were found in the samples of the same species treated with the different fixatives.

For transmission electron microscopy (TEM) investigations, the insects were fixed in 2.5% glutaraldehyde in 0.1 M Na–cacodilate buffer at pH 7.4 for 4 h at room temperature. The samples were then washed several times in the same buffer, post-fixed with 2% osmium tetroxide in the same buffer for 1 h at room temperature. They were then dehydrated in ethyl alcohol, immersed in propylene oxide and included in Embed 812. The ultrathin sections were collected on copper and rodium grids (200/300 mesh) and contrasted with uranyl acetate and lead citrate, according to Reynolds (1963).

Results

In this study the species Platycleis intermedia was used as a model; for the other species only the different characteristics, compared to the model species, will be reported.

I. Spermatheca of Platycleis intermedia (Serville)

Morphology and histology

The spermatheca is composed of a large seminal receptacle and a spermathecal duct. The seminal receptacle, sac-shaped, is 5 mm in length and 3 mm in width at its widest point. The spermathecal duct is tubular, winding, which when extended is about 2–3 mm in length with a calibre of about 0.3 mm.

In order to facilitate our investigation and description, the spermathecal duct was divided, for all the examined species, into three tracts. The distal tract nearest to the receptacle is called the connecting tract or ‘neck’, followed by the intermediate and the proximal tract, which opens into the vagina.

The general organization of the wall of the spermatheca is similar in the seminal receptacle and in the various tracts of the spermathecal duct. Histologically, from the internal to the external layer, it consists of a pseudostratified epithelium, surrounded by the cuticular intima, which delimits the lumen of the organ (Figure 1). The epithelium lies on a thin, fibrillar basal lamina and is delimited by a thick, connective-muscular sheath. These components gradually thicken from the distal to the proximal tract of the duct, with the consequent reduction of the organ lumen.

Figure 1. Platycleis intermedia, cross-section of seminal receptacle. c, cuticle; e, epithelium; L, lumen of seminal receptacle; mu, muscle. Haematoxylin–Eosin. Scale bar: 50 mm.

Since the epithelium of the seminal receptacle forms more or less prominent folds, its thickness is not constant along the same section. It can have a maximum thickness of about 53 µm. These folds reduce in thickness along the course of the spermathecal duct, until they completely disappear in the proximal tract.

The epithelia of the spermathecal duct, and that found at the level of the seminal receptacle, show a similar ultrastructural organization exhibiting two adjacent cell types: cuticle-forming cells and gland cells, which are not in contact with the intima (Figure 2).

Figure 2. Diagram of the ultrastructural organization of the epithelium of the seminal receptacle and spermathecal duct of Platycleis intermedia. 1, cuticle-forming cell; 2, gland cell. bl, basal lamina; ci, cuticular intima; g, Golgi bodies; gr, electron-dense granules; m, mitochondria; mt, microtubules; n, nucleus; r, reservoir; RER, rough ergastoplasmic reticulum; v, vesicle.

The cuticle-forming cells have a thin body and their nucleus, containing numerous chromatin granules, is elongated along the main axis of the cell; it is generally in a central or basal position.

The gland cells have the shape of a truncated pyramid with their oval or spherical nucleus in a basal position and containing few dispersed chromatin granules.

Ultrastructure

Seminal receptacle. The cuticle-forming cells and the gland cells are tightly packed (Figures 2, 3A). The contiguous membranes are rectilinear, with the exception of the apical region where they form deep interdigitations with septate junctions (Figure 3B, arrows). The cuticle-forming cells have a heterochromatic nucleus with numerous indentations, a perinuclear cistern with numerous dilations and the cytoplasm contains a rough ergastoplasmatic reticulum (RER) with dilated cisterns and numerous vesicles of varying size with heterogeneous content (Figure 3A). The Golgian areas are not clearly recognizable. The microtubules run parallel to the main axis of the cell both individually and grouped together (Figure 3C). In the apical region of these cells sac-like dilations are formed which contain a finely granular material, below which there are mitochondria of varying shapes (Figure 3D). Similarly to the spermatheca of other insects, the cuticular intima of Platycleis intermedia, about 4.5-µm thick, consists of an endocuticle with heterogeneous aspect and a thin granular epicuticle composed of an outer compact and intensely osmiophilic thin layer (about 47 nm) delimiting the organ lumen (Figure 3E, arrow).

Figure 3. Platycleis intermedia, seminal receptacle. A, the cuticle-forming cells (cc) and the gland cells (gc) are tightly packed. The perinuclear cistern appears dilated (arrow); RER, rough ergastoplasmic reticulum; n, heterochromatic nucleus; v, vesicles. B, the contiguous membranes form deep interdigitation with septate junctions (arrows); C, the microtubules (mt) in the cuticle-forming cells; D, the cuticle-forming cells around the apical region form sac-like dilations (little square). Inset: detail of the dilations in the little square; c, cuticle; gc, gland cell; gr, electron-dense granules; m, mitochondria; mi, microvilli of the end-apparatus; n, nucleus. E, apical region. Arrow: osmiophilic layer of epicuticle; end, endocuticle; ep, granular layer of epicuticle. F, gland cell. RER, rough ergastoplasmic reticulum is mainly made up of flattened and parallel cisterns. Scale bars: A, 92 nm. B, 1920 nm. C, 140 nm. D, 3390 nm. E, 480 nm. F, 870 nm.

The second type of cells or gland cells never reach the organ lumen; their large oval nucleus, in a basal region, is euchromatic and shows a clear nucleolus. The cytoplasm contains numerous elongated mitochondria and extended ergastoplasmic areas, which are mainly made up of flattened and parallel cisterns (Figure 3F). The Golgi complexes are not very evident. In the cytoplasmic region between the nucleus and the excretory pole, there is a high concentration of secretory granules with compact and electron-dense contents. The apical region displays a large invagination of the plasma membrane, which delimits the extracellular cavity known as the ‘reservoir’, which is more or less dilated in relation to the secretory activity. In correspondence to the ‘reservoir’ the plasma membrane forms numerous and prominent microvilli which are tightly packed and radially arranged around the cavity.

The reservoir possesses a single end-apparatus composed of microvilli and electron-dense material (felt-work) (Figures 3D, 4A). This material is made up of numerous fibrils, which run parallel to each other and perpendicular to the microvilli (Figure 4A). The cytoplasm surrounding the reservoir is particularly rich in mitochondria, electron-dense granules and electron-transparent vesicles. The efferent duct, of cuticular origin, opens into the reservoir and the material elaborated from the gland cells passes through this duct and is released into the lumen of the organ. The efferent duct is short, straight and perpendicular to the cuticular surface. The duct wall consists solely of epicuticle and is thinner at the reservoir level (Figure 4A, arrowhead), thickens immediately next to it (Figure 4A, arrow) and remains constant throughout its course.

Figure 4. Platycleis intermedia. A and B, seminal receptacle. A, the wall of the efferent duct consists solely of an epicuticle (ep) that is thinner at the reservoir (headarrow) and immediately after thickens and remains constant throughout its course (arrow); fw, felt-work. B, basal portion. Arrow, sediment of basal lamina; bl, basal lamina; e, epithelium; mu, muscular layer. C and D, connecting tract of the spermathecal duct: cuticle-forming cells. C, end, endocuticle; m, mitochondria; s, septate junctions. D, ae, apical expansions; v, vesicles with heterogeneous content. Scale bars: A, 650 nm. B, 1920 nm. C, 870 nm. D, 1510 nm.

All of the cells lie on a basal lamina surrounded by a thick connective-muscular layer (Figure 4B). The finely fibrillar basal lamina is mainly rectilinear, with some sediments which penetrate into the entire epithelial layer (Figure 4B, arrow).

Spermathecal duct. The general organization of the epithelium is similar to that of the seminal receptacle. The two cytotypes are narrower and higher; in their apical region, the contiguous membranes form deep interdigitations with wide systems of septate junctions (Figure 4C).

In particular, in the connecting tract, the cuticle-forming cells show some peculiarities. Some of them have irregular apical microvilli penetrating into the endocuticle, underneath which there are numerous mitochondria (Figure 4C); other cells, instead, have expansions variously extended with vesicles having heterogeneous content (Figure 4D).

The cuticular intima, which is thicker than that in the seminal receptacle, has an endocuticle with a strongly heterogeneous structure. Efferent ducts, with an essentially rectilinear course, pass through the cuticular intima and their content is granular.

The gland cells have an oval nucleus with small lumps of chromatin dispersed in the carioplasm. They also have a marked secretory activity, whereas this activity is low in the intermediate and in the proximal tracts. Furthermore, in the proximal tract the cuticle-forming cells have regular and tightly packed apical microvilli; the microtubule attachment plates (Figure 5A, arrows) are numerous between the contiguous cells and the endocuticle is strongly heterogeneous (Figure 5A). In this tract, the basal region of the cell, rich in mitochondria, form deep infoldings in which the basal lamina penetrates (Figure 5B, arrows).

Figure 5. A and B: Platycleis intermedia spermathecal duct. A, proximal tract, apical region of the cuticle-forming cells. Arrows, points of attachment of microtubules; end, endocuticle; mi, microvilli. B, basal region. Arrows, infoldings of the plasma membrane; bl, basal lamina; m, mitochondria. C–E: Platycleis grisea, seminal receptacle. C, gland cell. v, vesicles with heterogeneous content; D, vesiscles (v) with low electrondense content near to central reservoir (r); E, ed, efferent duct; v, vesicles with heterogeneous content. Scale bars: A, 1120 nm. B, 1920 nm. C, 870 nm. D, 870 nm. E, 650 nm.

II. Platycleis grisea (Fabricius)

The piriform seminal receptacle is 6.5 mm long and 3 mm wide at its widest point. The spermathecal duct has a constant calibre of 0.3 mm.

Ultrastructurally, the epithelium displays a general organization similar to that described above for Platycleis intermedia. In some gland cells, there are numerous vesicles with highly heterogeneous content (Figure 5C), probably of lysosomal origin. Vesicles, with low electron-dense content, are gathered in the cytoplasm near the central reservoir (Figure 5D). The content of the efferent duct is heterogeneous with electron-transparent vesicles of varying sizes (Figure 5E).

In gland cells, the RER is well developed throughout all the cytoplasm; it consists of dilated and flattened cisterns often parallel to each other.

The examined ultrastructural characteristics of the spermathecal duct are similar to those already described for P. intermedia.

III. Sepiana sepium (Yersin)

The spermatheca shows a piriform seminal receptacle, 3 mm long and 2 mm wide, and a tubular spermathecal duct, 3 mm long and 0.25 mm wide.

The wall of this organ has a pseudostratified folded epithelium; a cuticular intima, 3 µm thick, overlies the epithelium.

Ultrastructurally, the cytoplasm of the cuticle-forming cells is rich in free ribosomes, groups of microtubules, which run parallel to the main axis of the cell and vesicles of varying size with heterogeneous content. Numerous mitochondria are gathered in the apical area beneath irregular microvilli.

The cuticular intima consists of a fibrillar endocuticle and an epicuticle with small granules. It is surmounted by a thin, highly osmiophilic, layer.

The voluminous gland cells have a vesicular nucleus in the basal region, with few chromatin granules. The RER is widespread, the mitochondria are numerous and the Golgi complex is not evident. The reservoir is particularly dilated and delimited by sparse microvilli; its content is finely granular (Figure 6A). The cytoplasm around the reservoir displays vesicles with a finely granular material mixed with another more compact and electron-dense one (Figure 6A).

Figure 6. Sepiana sepium. A, seminal receptacle. Arrow, cuticle; ed, efferent duct; L, lumen; r, reservoir; v, vesiscles with heterogeneous content. B, proximal tract of the spermathecal duct. Cuticle-forming cells. Arrow, osmiophilic layer of the epicuticle; end, endocuticle; r, reservoir. C, arrows, break of the osmiophilic layer of the epicuticle; L, lumen; sp, sperms. D, the cytoplasm of the gland cells has numerous vesicles (v) with a low electron-dense content. E and F Tessellana tessellata, seminal receptacle. E, vesicles (v) in the cytoplasm around the reservoir (r); ed, efferent duct; m, mitochondria; n, nucleus. F, gland cells. g, Golgi bodies; r, reservoir; v, vesicle with a granular content opening into the reservoir. Scale bars: A, 1510 nm. B, 1920 nm. C, 1120 nm. D, 1920 nm. E, 2000 nm. F, 480 nm.

The rectilinear efferent duct displays a content consisting of very thin, irregular and tightly packed fibrils. A similar material is found inside the lumen of the organ near the cuticle (Figure 6A, arrow).

The general ultrastructural organization of the spermathecal duct is not substantially different from that of the seminal receptacle.

However, in the connecting tract, some areas have peculiar ultrastructural characteristics involving both the epithelium and the upper cuticular intima. In these areas, the cuticle-forming cells undergo partial or total lysis: their cytoplasm is highly vacuolized, the apical microvilli are reduced or completely absent. The cuticular intima loses its typical organization except in the highly osmiophilic superficial layer of the epicuticle (Figure 6B, arrow). The epicuticle is open in several tracts and folds back towards the epithelium (Figure 6C, arrows). The lumen is rich in isolated, often altered, sperm (Figure 6C). Furthermore, in these regions, the cytoplasm of the gland cells has numerous vesicles, which vary in size and have a low electron-dense content (Figure 6D).

IV. Tessellana tessellata (Charpentier)

The sac-like seminal receptacle is about 5 mm long and 2 mm wide. The cuticular intima is 3–4 µm thick. The rectilinear spermathecal duct is about 2 mm long.

From the TEM observations, the cytoplasm of the cuticle-forming cells appears rich in free ribosomes and vesicles with an electron-dense content.

The microtubules are grouped together and run parallel to the main cell axis. The numerous mitochondria are more abundant in the apical region, under the microvilli (Figure 6E).

The cuticular intima has a fibrillar endocuticle with fibrils loosely arranged near the microvilli and its epicuticle, containing very small granules, is surmounted by a very thin irregular and highly osmiophilic layer, as in the other species.

The gland cells have a voluminous nucleus, in the basal region, with few chromatin granules and an evident nucleolus. In the apical portion of the cell there is a very dilated reservoir surrounded by long microvilli (Figure 6E). The cytoplasm around the reservoir is rich in vesicles, with a granular content, some of which seem to open into the reservoir between the microvilli (Figure 6F).

The RER is in the paranuclear zone, where it forms several flattened parallel cisterns. The Golgi complex is evident and consists of sacculi and vesicles, with a low electron-dense content (Figure 6F). The secretion of the rectilinear efferent duct is granular and electron-dense (Figure 6E).

The epithelial cells lie on a thin basal lamina consisting of packed fibrils, which penetrates deeply between the contiguous cells.

There are no substantial differences in the ultrastuctural organization of the three tracts of the spermathecal duct between T. tessellata and P. intermedia. However, in the connecting tract, as described for Sepiana, the cuticle-forming cells have an extended rough endoplasmatic reticulum and marked secretory activity. None the less, many of these cells show clear signs of degeneration as evidenced by the highly vacuolized cytoplasm and by the reduction or complete absence of the apical microvilli. The cell boundaries between the cuticle-forming cells and the gland cells are not often easily distinguishable.

Discussion

Anatomically, our observations show a relatively uniform morphology of the seminal receptacle and the spermathecal duct of the spermatheca, in agreement with data present in the literature for other species belonging to Orthoptera Ensifera.

The general morphological homogeneity of the spermatheca of the Tettigoniidae examined is also accompanied by histological and ultrastructural uniformity; the wall of this organ gradually thickens in the spermathecal duct. The epithelium consists of two different cytotypes: the cuticle-forming cells and the gland cells. However, in the spermatheca of other species of Ensifera examined, Gryllus domesticus, Liogryllus bimaculatus (Gryllidae) (Sathe & Joshi 1988) and Dolicopoda schiavazzi (Melis & Dallai 1963), the epithelium consists only of cuticle-forming cells; in the genus Gryllotalpa (Grillotalpidae) (Melis & Dallai 1966; Sathe & Joshi 1988), elements specialized in the gland function in same tracts of the spermathecal duct were observed, whereas the epithelium of the seminal receptacle consisted only of cuticle-forming cells.

Further differences between the above-mentioned Orthoptera Ensifera and our species revealed a more efficient division of the functions of the epithelial cells: the gland cells elaborate the spermathecal secretion, whereas the cuticle-forming cells mainly perform the secretion of the cuticular intima and of the epicuticle of the efferent ducts.

In some Orthoptera Acrididoidea and Phasmatodea (Viscuso et al. 1994) this latter function is carried out by a third cytotype in the spermathecal epithelium: the ‘duct cell’. The duct cell is considered a specialized cuticle-forming cell due to its cytological characteristics and functions (Noirot & Quennedey 1974; Quennedey 1998). Instead, in the Tettigonioidea and Grilloidea, the spermathecal epithelium lacks any other further specializations of the cuticle-forming cells; thus suggesting a greater primitiveness of these groups with respect to Acridoidea.

In the species we examined, the gland cells are never in contact with the cuticular intima and their general organization is identifiable as class 3 according to the Noirot and Quennedey (1974) subdivision regarding the ectodermic glands of insects.

The secretion elaborated by the gland cells differs in its aspect between different species. Only in P. intermedia does this secretion consist of uniformly compact and electron-dense granules.

Secretion inside the reservoir seems to take place by the fusion of the vesicles with the membrane, which delimits the reservoir; this is partly similar in Periplaneta (Gupta & Smith 1969).

Both invaginations of the basal plasma membrane of the epithelial cells and numerous mitochondria, in the terminal tract of the spermathecal duct, might imply the involvement of these cells in the re-absorption process (probably from the haemocoel), as already noted in other insects (Blum et al. 1962). However, it is also possible that the invagination systems, in the species we examined, could be correlated more with the ionic transport from the haemocoel, as already referred in other tissues of insects (Anderson & Harvey 1966; Berridge & Oschman 1969), rather than the re-absorption of macromolecules.

Ultrastructural analysis shows that the gland cells of the spermatheca, in the same specimen, can have different functional stages, above all if, on the one hand, we consider the gland cells of the connecting tract of the spermathecal duct (which are usually more active) and, on the other hand, the gland cells of the other duct tracts that do not show signs of any marked synthesis activity. However, this does not exclude the fact that in the latter tracts the gland cell activity, which is usually limited, can increase at specific functional moments (for example, when sperms are transported, etc.).

The general structural organization of the cuticle-forming cells is similar to that seen in other insects. Throughout the spermathecal tracts, the presence, in the apical region, of mitochondria that are grouped beneath numerous microvilli, suggests that these cells have a re-absorption role. Similar cytological aspects already seen in the epithelial cells of the spermatheca in Periplaneta americana (Gupta & Smith 1969), Tenebrio molitor (Happ & Happ 1975), Apis mellifera (Dallai 1975) and Melanoplus sanguinipes (Ahmed & Gillot 1982), seem to be characteristic of cells involved in the ionic transport process (Berridge & Oschman 1972). For example, in A. mellifera, between the cells of the spermathecal wall and the lumen of the organ, there is a different ionic concentration maintained by active transport (Gessner & Gessner 1976).

In the cuticle-forming cells, groups of microtubules, perpendicular to the surface of the organ, assure resistance to mechanical strain of the epithelium during the transit of the gametes. Moreover, in addition to the cytoskeletric system, this role may be carried out by numerous junction systems, such as cellular interdigitation and septate junctions in the lateral plasma membrane.

The cuticular intima of the spermatheca could carry out different functions: first of all, it constitutes of a type of ‘endoskeleton’ which supports the organ, keeping the lumen of the various tracts open; it could also have a function of ‘isolating’ the spermathecal epithelium from the secretions in the lumen, preventing cells coming into direct contact with them.

The last function of the intima is of particular importance in some cases; in the Acridoidea, for example, the lytic activity, carried out by the spermathecal secretions, is used for the demolition of the spermatophore, transiting in the spermatheca, and of the spermatozoa when, inside the seminal receptacle, they undergo a process of remanagement, with their partial or total demolition (Longo et al. 1993). However, in the Acridoidea examined, this lytic activity does not involve any direct participation of the wall cells of the organ, unlike what has been noted in the spermatheca of Rhacocleis annulata (Tettigoniidae), where the activity of lysis of the sperm occurs in the connecting tract of the spermathecal duct, in a progressive sequence of events primarily involving the cells of the wall (Viscuso et al. 1996a).

In the species here examined, the ultrastructural aspects of the connecting tract in the spermathecal duct show a secretory activity of the gland cells, which is more marked than in other tracts of the duct. In particular, in T. tessellata, the secretory activity of the cuticle-forming cells and the gland cells is accompanied by an evident cytoplasm vacuolization; even in S. sepium, peculiar aspects of lysis of the epithelium and the cuticular intima have been seen, largely similar to what has already been described for the same region in R. annulata (Viscuso et al. 1996a).

It is known that in the genital tracts of various insect species the high secretory activity of some cells is often accompanied by a cytoplasmic vacuolization (Hamon et al. 1982; Viscuso et al. 1985, 1996a, 1996b, 2005), which can precede the lysis of these cells (Riemann 1973; Viscuso et al. 1985). Nevertheless, when this lysis seems to involve different parts of the genital tract at the same time, it could indicate the end of the reproductive period of an individual. In the species examined, instead, vacuolization of the epithelial cells was constantly found in a specific region of the spermathecal duct (connecting tract) where, in some cases, as was stated before, also a lysis activity was found.

Taking this into consideration, the lack of evidence of lytic activity in the same tract of the other species examined, where the secretory activity is even more marked, could be related to the functional moment.

It is on the basis of this evidence and these considerations that we could hypothesize, as for R. annulata, that in the connecting tract of the duct, at least in some Tettigoniidae, there is a lysis activity of the epithelium aimed at the rapid capture and consequent lysis of the surplus of spermatozoa transferred by the male during copulation.

However, lysis could also be a mechanism making room for sperm from a new mating; in insects, which copulate repeatedly, in fact, the sperm from the most recent mating are biologically advantageous for egg fertilization (Walker 1980).

The above-mentioned hypothesis is also supported by the remark that in the Orthoptera Tettigoniidae there is no specific anatomical structure comparable to a fertilization chamber able to collect the spermatozoa that will fertilize the egg and from where they would be eventually removed before further mating, as, however, was found in other insects such as Diptera (Marchini et al. 2001). The anatomical situation of the Tettigoniidae could thus justify the presence of a specific tract of the spermathecal duct that is able to regulate spermatic material.

A lysis activity involving both epithelium and cuticular intima is already known in the genital tracts of other insects: in the spermatheca of the Apidae Melipona bicolor; indeed, this activity has been related to a spermiophagic function performed by some cells of the organ wall (Da Cruz-Landim 2002). A lysis activity of the epithelium which involves the cuticular intima has also been described in Isopoda Oniscidea: in the oviduct of Porcellio laevis, indeed, the epithelium and the cuticular intima undergo lysis phenomena due to a secretory activity of the oviduct glands; this lysis activity facilitates egg release from the ovary and sperm entrance into the seminal receptacle (De Luca et al. 1987; Longo et al. 1998).

Recent research has shown that, in the Tettigoniidae, the feather-shaped spermatodesms from the spermatophore with the male accessory gland secretions are sent to the seminal receptacle where they are stored inside the spermatodoses (Viscuso et al. 2002). These are small, sclerified capsules with a particular morphology depending on the species (Viscuso et al. 2002; Vahed 2003), which are found within the seminal receptacle, following and/or at the same time as the transfer of the male gametes.

Even if there is a lack of demonstrative data on the genesis of the spermatodoses, Vahed (2003) has hypothesized that the secretion of the male accessory glands is responsible for its genesis. Although we agree with this hypothesis, nevertheless it is probable that the secretion from the spermathecal epithelium also contributes to the genesis of the spermatodoses.

In conclusion, from the results obtained it would seem reasonable to hypothesize that, in the examined species, the seminal receptacle and the spermathecal duct are different, from the functional point of view, despite showing similarities in their structural organization. Indeed, only the spermathecal duct and, in particular, the connecting tract would appear to control and regulate the gametes transferred by the male during copulation in order to confine fertilization activity exclusively to spermatozoa within the seminal receptacle.

Instead, the seminal receptacle would be predominantly responsible for the genesis of spermatodoses, for their storage and, at the same time, it could play a role in the release of single sperms from the feather-shaped spermatodesms, before they interact with the female gamete.

Acknowledgements

This research was supported by a MIUR grant, Scientific research programs, co-financing: ‘Structural and molecular studies on insect reproduction’.