Anatomy, Histology and Function of the Reproductive System of the Tracheopulmonate Slug Athoracophorus Bitentaculatus (Quoy and Gaimard).
The reproductive system of Athoracophorus is described in detail from serial sections and found to be basically similar in structure and histology to those found in species from other families of terrestrial slugs. The term "oviducal gland" is substituted for the terms "glomerate gland" and "uterus" previously used, and the reasons for this are discussed. Functional aspects of the system are examined.
Until recently, the New Zealand and Subantarctic Athoracophoridae have been a somewhat neglected group. Most workers on the Athoracophoridae have confined their attention to taxonomy and anatomy, and little has been paid to the finer details of their organisation, or to their ecology, development, feeding habits, breeding behaviour, and genetics. There are, of course exceptions, one of the more notable being a contribution by Plate (1898), who investigated the structure of the dorsal tracheate lung and of the renal system in some detail. However examination of reproductive systems has always been limited to dissection alone.
Reproductive systems of a number of species of New Zealand Athoracophoridae have been described by Hutton (1881), Plate (1898), Suter (1913), Burton (1962, 1977), and Climo (1973). The most obvious differences in anatomy between the various species lie in the relative arrangement of the prostate gland (referred to as the bulbose gland by Suter), the uterus, the spermatheca, and the oviducal gland. The naming of the oviducal gland has caused considerable confusion in the past; it has been variously referred to as the glomerate gland by Suter (1913) and Burton (1962, 1977), as the shell gland ("schalenblaschen"—literally shell bubble) by Plate (1898), and as the uterus by Climo (1973). Names put forward for a similar structure in other families of terrestrial slugs include "female gland" in Arion ater (Smith, 1966), "nidamental gland" in Philomycus carolinianus (Kugler, 1965) and "oviducal gland" in Agriolimax reticulatus (Boyne, 1966). Out of this nomenclatural plethora the term "oviducal gland" was finally selected for two reasons. First, the reproductive system of Athoracophorus bitentaculatus appears very similar in structure and function to that of Agriolimax reticulatus. Second, the lumen of the gland does form the oviduct; the uterus is a much more muscular structure, and quite different in function.
The present study describes the form of the reproductive system and some of its histological features, and the object of the paper is to provide a foundation on which to base further studies on the histochemistry and function of the reproductive systems of the Athoracophoridae, and on their breeding cycles.
Material and Methods
Adult specimens of the slug Athoracophorus bitentaculatus were collected from the leaf bases of a flax plant (Phormium tenax) in Ngaio, Wellington, from March to September. The animals were narcotised in a relaxed state in mentholised water according to the method of Abdel-Malek (1951). The entire reproductive system was dissected free with the aid of a binocular microscope, fixed in Bouin's solution for three hours, placed in a saturated solution of lithium carbonate in 70% isopropanol for 1 hour, and stored in 70% isopropanol until required. After dehydration, the material was placed in chloroform overnight rather than in xylol, as it was found that clearing in xylol, even for periods as short as 30 minutes, rendered the tissues of the albumen gland very hard and brittle. The entire reproductive system was then embedded in 56°C —melting point paraffin wax and serially sectioned at 7 μm. Sections were stained in either Ehrlich's haematoxylin and alcoholic eosin, or in Masson's trichrome stain, or with PAS, using Delafield's haematoxylin as a nuclear stain.
To facilitate the construction of a sectional diagram of the reproductive system, every 6th section was photographed, and relevant portions of the photographs were traced to build up a composite picture.
The reproductive system of the slug Athoracophorus bitentaculatus consists essentially of a gonad capable of producing eggs and sperm simultaneously, and a compound tubular passageway through which the sperm and eggs are conveyed externally. Three different major glands feed into this passageway; these are the albumen gland, which secretes a nutrient solution around each egg, the oviducal gland, which secretes a number of encapsulating mucus coats around each egg, and the prostate gland, which produces a secretion of unknown function in which the sperm are immersed. Portions of the passageway are lined with cilia, and the terminal portions are modified into copulatory apparatus. A cutaway diagram to show the relationship of the various structures is given in Figure 1.
The gonad consists of several lobes, closely fitted together. Each lobe consists of a number of tubular acini, each acinus being broadest at the surface of the gonad, and narrowing down to a small efferent ductule which communicates with the lumen of the hermaphrodite duct. The hermaphrodite duct is quite long, highly convoluted and of constant diameter along most of its length but narrow close to the albumen gland; it passes from the ovotestis downward and anteriorly to the medial surface of the albumen gland, which is compact, elongate, medially flattened and yellow in colour. The secretion of this gland passes into an albumen canal, embedded in gland tissue and lying close to the medial surface of the gland. The canal communicates directly with the hermaphrodite duct to form an irregularly shaped chamber, the fertilisation sac.
Fig. 1: Cutaway diagram of reproductive system, ventral view. Vertical component not to scale. The connection between the fertilisation pocket and the female duct is out of the plane of section, and is shown dotted.
Abbreviations: a. gl., albumen gland; f.p., fertilisation pocket; g., gonad; h. d., hermaphrodite duct; ov. gl., oviducal gland; p. gl. prostate gland; p.r.m., penis retractor muscle; pen., penis; pen. s., penis sheath; sp., spermatheca; u., uterus; vag., vagina; v. d., vas deferens.
The lumen of the uterus carries extensive longitudinal ridges, which persist to the point at which the spermathecal duct enters the uterus; at this point the ridges become transverse. The spermatheca is moderately thin-walled, rounded, and attached to the anterior end of the uterus on the dorsal side by a short, tubular stalk, the opening of which is guarded by valves. Immediately anterior to the insertion of the spermathecal duct, the uterus makes a Y-shaped junction with the penis, laterally, and the vagina, anteriorly.
The penis of Athoracophorus bitentaculatus is basically a muscular convoluted cylinder, contained in a penis sheath, and narrowing distally to join the vas deferens. At the junction with the vas deferens, the penis sheath merges with the penis, and a penis retractor muscle is inserted. The origin of the penis retractor muscle lies in the body wall on the dorsal side posterior to the mantle area. The lumen of the penis is heavily ridged and papillated.
From its junction with the penis at the point of insertion of the penis retractor muscle, the vas deferens runs anteriorly over the surface of the penis to pass dorsally over the uterus down the anterior wall of the spermatheca, and then posteriorly to pass through the tissue of the anterior portion of the prostate gland to join the spermoviduct.
The vagina extends anteriorly from its junction with the penis and uterus to open at the genital orifice situated in the right margin of the head shield, just lateral to the right tentacle.
The gonad consists of a small number of lobes, each lobe comprising numerous acini. Connective tissue septa, containing blood vessels, separate the acini and merge into efferent ductules lined with low cuboidal ciliated epithelium at the bases of the acini. In mature gonads, the walls of the acini are lined with clusters of germinal epithelial cells, and the acini contain both ova and sperm. The ova are always situated peripherally. Each ovum adheres to the wall of the acinus with its free side directed towards the lumen and covered with thin layers of cells apparently derived from the germinal epithelium. As the ovum grows, a cavity appears between this follicular membrane and the developing egg (Fig. 2).
Like the eggs, the sperm arise from cells of the germinal epithelium of the acini. Each male germinative cell develops into a cluster of spermatogonia in which all members undergo a synchronous development (Fig. 3). The cluster apparently becomes attached to a group of germinal epithelial cells, and is arranged with the sperm heads directed towards the periphery of the acinus, and the sperm tails trailing into the lumen (Fig. 4).
In immature specimens, no ova can be seen, although numerous developing sperm may be present (Fig. 5).page 5
Fig. 2: Cross section of an oocyte fairly late in development, showing the cavity which develops between the oocyte and the follicular cells. Masson's trichrome stain.
Fig. 4: Cross section of gonad. Sperm are arranged in clumps with heads close to the acinar wall, and tails trailing into the lumen of the acinus. Haematoxylin/eosin.
The tubular, convoluted hermaphrodite duct has a two-layered wall, composed of a thin outer connective tissue sheath closely applied to an inner layer of simple, low columnar ciliated epithelium (Fig. 6). The epithelial cells become taller as the duct nears the albumen gland, and the duct narrows appreciably. In mature animals taken during autumn, the duct is packed with sperm for most of its length, only the end near the albumen gland being clear (Fig. 7). A few scattered flask-shaped mucus cells are present in the epithelial wall.page 7
Fig. 6: Cross section of hermaphrodite duct, with ciliated cuboidal epithelium, and a packed mass of sperm in the lumen. PAS stain.
The albumen gland is a compound tubular gland, the walls of the tubules being composed of large apocrine gland cells with basal nuclei. These cells contain numerous large ovoid granules, which stain a pale violet colour in haematoxylin/eosin preparations. Scattered granules stain positively with PAS (Fig. 8). A thin layer of squamous epithelium lines the tubules, which are bound together by thin sheets of connective tissue. The tubules of the albumen gland empty their transparent, nutritive secretion into the albumen canal which runs the length of the gland, and is also thinly lined with squamous epithelium. The albumen canal joins the hermaphrodite duct to form a thick-walled chamber, the fertilisation pocket (Fig. 9).
The simple columnar ciliated epithelium of the hermaphrodite duct undergoes an abrupt transition to a thick, pseudostratified, ciliated epithelium with a minutely lobulated surface upon reaching the fertilisation pocket. At the level of entry of the hermaphrodite duct and albumen canal, the pocket is U-shaped in cross section, but expands dorsally to form an irregular chamber with thick walls composed of irregularly-shaped cells with basal nuclei and minutely granulate cytoplasm. A thick connective tissue sheath supports the fertilisation pocket, and strands of connective tissue pass through its walls. Close to the origin of the sperm duct the pocket is lined by ciliated cuboidal epithelium containing a few scattered large goblet cells.
The walls of the male duct as it emerges from the fertilisation pocket are composed of ciliated low simple columnar epithelium, supported by a thick outer layer of connective tissue. As the male duct passes anteriorly over the folds of the prostate gland, receiving ciliated ductules from the prostate gland at several points (Fig. 12), the epithelium of the wall gradually changes to simple ciliated cuboidal epithelium. Subepithelial mucus-secreting cells are present in the wall. The duct varies in section from a deep cleft to a round tube, and in the anterior section it is in open communication with the lumen of the oviducal gland to form a spermoviduct (see Fig. 11).
Female duct and oviducal gland. (Fig. 10)
The female duct forms the lumen of the extensive, broadly convoluted oviducal gland, and is irregular and capacious. It communicates directly with the sperm duct near its anterior end. The oviducal gland is mainly composed of long, relatively narrow cells, with a central irregular nucleus and very pale cytoplasm; a few interspersed smooth muscle cells can also be seen. Connective tissue sheaths the gland, and the lumen is lined with a thin layer of simple squamous epithelium. No cilia are present. Near the bifurcatio spermoviducti the gland cells are somewhat smaller, the epithelial lining of the lumen considerably thicker, and more connective tissue is present. The mucus-secreting cells which make up the oviducal gland do not cease at the bifurcation, but continue for a short distance into the uterus as a subepithelial layer.page 9
Fig. 8: Section of albumen gland of mature specimen, showing strongly PAS-positive granules (arrowed).
Fig. 9: Section through fertilisation pocket. The hermaphrodite duct (arrowed) joins the fertilisation pocket close to its junction with the albumen canal. Haematoxylin/eosin.
The compound tubular prostate gland is largely composed of tall pyramidal cells with rounded basal nuclei and densely, minutely granular cytoplasm. A few very narrow interstitial cells with elongate nuclei can be seen between the secretory cells. The tubules of the gland are held together by thin sheets of connective tissue, and have a narrow, ciliated lumen communicating directly with the male duct (Figs. 11 and 12).
As the vas deferens diverges from the spermoviduct its epithelial lining is composed of tall, ciliated columnar cells with prominent elongate vacuoles. The cilia disappear a short distance down the vas deferens, but otherwise the appearance of the cells remains the same along the length of the tube. A thin layer of connective tissue sheaths the vas deferens (Fig. 12).
As the uterus passes from the spermoviduct, the epithelium is thrown up into extensive longitudinal folds. The simple, ciliated, columnar epithelium rests on a layer of connective tissue, which is enclosed by a heavy investment of circular muscle, with a thin layer of longitudinal muscle forming the outside of the tube. The uterus spirals forward to a Y-shaped junction with the penis and vagina, and receives the spermathecal duct just posterior to this junction. A heavy layer of mucus coats the uterine wall.
Spermatheca and spermathecal duct.
The spermatheca is simply a large two layered sac, sheathed by a thin layer of connective tissue and lined by simple non-ciliated, low columnar epithelium. The epithelial cells contain large vacuoles similar to those in the epithelium of the vas deferens. As the wall of the spermatheca merges into the spermathecal duct, the cells of the epithelial lining become taller and the connective tissue thicker. Smooth muscle invests the walls of the duct, and the duct's entrance into the uterus is guarded by valves.page 11
Fig. 11: Section through prostate gland and developing oviducal gland of immature specimen, showing the relationship between the male and female grooves. Masson's trichrome stain.
As the vas deferens nears the distal end of the penis, its wall becomes thickened by a dense layer of circular muscle. This layer merges into the penis sheath, and becomes separated from the penis proper by a fluid-filled space. No longitudinal muscle fibres are present in the sheath. Inside the sheath, the heavily convoluted and folded penis is lined by ridged columnar epithelium resting on connective tissue and surrounded by an inner circular and an outer longitudinal muscle layer. Thick mucus lines the lumen of the penis. As the penis nears its junction with the uterus and vagina, the folding and ridging of the epithelium becomes more prominent, and the muscle layers thicker. The penis sheath merges with the penis at the junction with the uterus and vagina (Fig. 14).
The vagina is similar in structure to the uterus but the muscle layers of its walls are thinner.page 13
Published accounts of the reproductive systems of various species of New Zealand Athoracophoridae generally agree fairly closely. Most accounts show the prostate and oviducal glands as discrete structures, with only minor differences in morphology between the species. An exception is Pseudaneitea ramsayi (Climo, 1973) in which the prostate gland and the oviducal gland (called the uterus in Climo's description) form an elongate, intertwined, irregular complex up the central axis of the reproductive system. A similar pattern can be seen in the reproductive system of Athoracophorus bitentaculatus and this arrangement is quite different from that seen in Pseudaneitea papillata, for example (Burton, 1962). It is still not clear whether a basic difference in the form of the reproductive system does exist, or whether it is due to seasonal or growth factors.
Athoracophorus bitentaculatus, like other pulmonate Gastropods, is hermaphroditic, and has a complex reproductive system. This system appears to be capable of producing both eggs and sperm for a considerable period of the year, at least from Autumn through Winter to Spring. It is highly likely that a slug will mate a number of times through the breeding season. When this mating occurs, a number of events take place within the reproductive system to facilitate it.
In the male system, sperm produced by the gonad are stored in the hermaphrodite duct, which becomes distended with sperm, except for the portion nearest the albumen gland. The passage of sperm through the fertilisation sac, down the male duct, and into the vas deferens and penis is obviously not a continuous process, as no sperm were found in any of these organs. Such a movement of sperm through the reproductive tract probably occurs only just prior to copulation, or during copulation itself. While slugs have been observed in copulo, no courtship behaviour patterns have ever been recorded in the Athoracophoridae. However, elaborate courtship sequences have been described for many slugs, and it seems that one function of courtship is synchronisation of the physiology of the mating partners (Runham and Hunter, 1970). It seems highly likely that such a courtship sequence occurs in the Athoracophoridae as well.
The movement of sperm through the reproductive tract is probably passive, as all ducts are either ciliated (hermaphrodite duct, male duct, vas deferens) or muscular (penis). This movement of sperm is aided by secretions from various sources, notably the prostate gland and the vas deferens. The prostate gland secretes the seminal fluid, the function of which is uncertain. It is generally believed to be nutritive (Kugler, 1965), although it contains a number of non-nutritive constituents whose function is unclear (J. M. Cummins, pers. comm.). In Philomycus carolinianus, subepithelial glands of the spermatic groove produce mucus which binds the sperm together into a sperm thread. Similar subepithelial mucus glands are present in Athoracophorus bitentaculatus.page 15
In summary, the sperm is produced by the gonad, stored in the hermaphrodite duct, probably until the onset of courtship, passed by ciliary action through the fertilisation sac and along the spermatic groove to be bound by mucus secretions into a sperm thread, then reaches the vas deferens at the bifurcatio spermoviducti, and passes down into the eversible penis for insertion into the vagina of the mating partner. Once inserted into the vagina, the sperm mass enters the spermatheca, and is apparently stored there until required. The exact mode of sperm transfer into and up the duct of the spermatheca is not known, but the penis would certainly extend as far as the opening of the duct when fully inserted into the vagina, and it is conceivable that the penis could inject the sperm mass directly into the spermatheca. Holm (1946) in studies on Lymnaea stagnalis appressa Say, observed that in comparatively young snails, nearly all the sperm transferred at the time of copulation passed into the receptacle, and few sperm were seen in the upper vagina. Furthermore, the sperm in the spermatheca of Athoracophorus bitentaculatus is, for the most part, still in a compact, mucus-bound mass, and it seems unlikely that they could swim unaided into the receptacle.
Ikeda (1937) determined that in the slug Meghimatium bilineatus the spermatophores received during copulation were retained for a period in the genital atrium, where the sperms were loosened from the sperm mass, and lost their tails. The tailless sperms were then passively conveyed to the spermatheca and stored until oviposition. No tailless sperm were observed in Athoracophorus bitentaculatus and this agrees with the finding of Kugler (1965) for the slug Philomycus carolinianus. In A. bitentaculatus, the sperm mass retained within the spermatheca tends to break up under the influence of the secretions of the spermathecal wall. It is clear that sperm may be moved passively up to the point at which they enter the spermatheca, through either ciliary action or muscular movement. However, the lumen of the uterus is thickly coated with mucus, and its walls are non-ciliated. Presumably, the sperm swim unaided up the uterus to the fertilisation pocket, and if this is the case it follows that the function of the secretions of the spermathecal wall may be twofold, in that they loosen the sperm from the sperm mass and induce sperm capacitation.
The female system is functionally less complex. In pulmonates in general, there is a continuous production of ova, sperm and nutritive cells in the mature gonad, except towards the end of the breeding season (Runham and Hunter, 1970). Smith (1966) observed that in Arion ater maturing gonads commence sperm production before ova appear, and this is also the pattern in Athoracophorus bitentaculatus. However, A. bitentaculatus does not show the clear separation of the male and female phases of reproduction seen in Arion ater, and thus conforms more closely to the generally accepted pulmonate pattern.
The egg is considerably larger than the efferent ductule. However, the efferent ductule is heavily ciliated and, like the hermaphrodite duct, is apparently capable of some distention. As the egg is oval, passage of the egg through the narrow efferent ductule probably presents little difficulty. The hermaphrodite duct is normally packed with sperm, but as copulation takes place some time before egg laying, sperm would be cleared from the hermaphrodite duct in time to allow the passage of the ova for fertilisation in the fertilisation pocket. The fertilised ova then page 16receive the nutritive secretion from the albumen gland, and pass to the oviducal gland to receive a layered, gelatinous shell. The eggs then pass down the uterus to be laid in clusters of 15 to 20; they are round, papillate, gelatinous, light yellow, and up to 3 mm. in diameter.
Overall the organisation of the reprodutive system of Athoracophorus bitentaculatus shows a striking similarity to that of other slugs, such as Agriolimax reticulatus and Arion ater. Some differences do occur, such as the enclosure of the penis in a penis sheath and the partial separation of the male and female ducts, but the organisation and histology of the reproductive tract of these species is similar enough to underline once again the conservativeness characteristic of the Mollusca.
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