07 197-204 Gomez 31/3/0 10:59 am Page 197 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositório da Universidade dos Açores J. Moll. Stud. (2000), 66, 197–204 © The Malacological Society of London 2000 CALCIUM PHOSPHATE GRANULES IN THE REPRODUCTIVE SYSTEM OF OXYCHILUS ATLANTICUS (GASTROPODA: PULMONATA) B.J. GOMEZ1 and A.S. RODRIGUES2 1Departamento de Zoología y Dinámica Celular Animal. Facultad de Ciencias. Universidad del País Vasco. Apdo 644. Bilbao 48080. Spain. 2Departmento de Biologia. Universidade dos Açores. Rua da Mae de Deus, 58. Apart. 1422. 9502 Ponta Delgada. Açores. Portugal (Received 7 June 1999; accepted 8 September 1999) ABSTRACT Characterization of accumulating cellular types in molluscs, as well as their physiological The presence of connective interstitial cells in the wall role, was first described by Fournié & Chétail of several organs of the distal reproductive system in (1982). According to these authors there are Oxychilus atlanticus is described by means of scan- four accumulating calcium cellular types in ning electron microscopy (SEM). Connective inter- stitial cells extend from the distal part of the vas molluscs: free connective cells, secretory sub- deferens to the penis, being the dominant cell type of tegumentary cells, digestive gland calcium cells, large portions of the epiphallus as well as in the whole and amebocytes. penial caecum (ϭflagellum). These cells are highly Various forms of connective interstitial cells mineralized and are characterized by the presence have been histologically described in the wall of of large amounts of solid spherules (up to 4 ␮m in the male reproductive system distal ducts in diameter) in their cytoplasm. A semi-quantitative several stylommatophorans. Lusis (1961) and evaluation by X-ray microanalysis demonstrates that Smith (1965) working on Arion rufus (L.) and calcium and phosphorus are the main elements of the Arion ater (L.), respectively, describe them as spherules. Their function is unknown, but they could aid in copulation or they could assist the survival of vacuolated cells. Also in several helicids such as the transferred spermatozoa. Helix pomatia Linnaeus and Theba pisana (Müller) interstitial round cells, which stain light with general histochemical methods have INTRODUCTION been identified in large amounts in the connec- tive wall of the epiphallus (Noyce, 1973). They Oxychilus (Drouetia) atlanticus (Morelet and have also been recognized in the epiphallus of Drouët, 1857) is a stylommatophoran snail of Arion subfuscus (Draparnaud) (Zubiaga, 1986) the Family Zonitidae, endemic to São Miguel and Arion hortensis Fèrussac (Walls, unpub- Island, Azores. Several systematic studies have lished in Runham, 1988). Histochemically, these been undertaken on this species (Martins, 1991; vacuolated cells have been shown to contain Brito, 1992), mainly due to the great inter- non-sulphated acidic polysaccharides (Smith, specific variability. Presently, we are studying 1965; Zubiaga, 1986), but no positive reaction the structure and function of the reproductive has been obtained for calcium demonstration. system (Rodrigues, Gómez, Cunha & Martins, Most of these interstitial cells have been 1998; Rodrigues & Gómez, 1999) in order to described as secretory cells. Only for Arion advance in the knowledge of the reproductive hortensis they have been described as connec- biology of this species, a potential predator of tive tissue cells containing large spherules Lymnaea truncatula (Müller), the intermediate resembling calcium granules (Runham, 1988) host of the trematode Fasciola hepatica (L.) resembling those we have found in Oxychilus (Cunha, 1991). During the course of these atlanticus. The presence of similar granules has investigations we have observed the presence of also been indicated in several reproductive connective interstitial cells in the distal ducts of organs of Arion rufus (Lusis, 1961) such as the the reproductive system of O. atlanticus. These bursa copulatrix, the hermaphrodite duct, the cells are characterized by the accumulation of vas deferens, the oviduct, and the atrium, and great amounts of spheroid granules in their described as small amounts of large spherules, cytoplasm. measuring up to 8 ␮m in diameter. 07 197-204 Gomez 31/3/0 10:59 am Page 198 198 B.J. GOMEZ & A.S. RODRIGUES As far as we know the chemical composition the copulatory process there is both a progres- of such granules has never been investigated. sive eversion of penis and intromission of this Scanning electron microscope energy-dispersive organ into the female ducts until the everted X-ray microanalysis has been undertaken in the penial caecum reaches the bursa copulatrix present work to determine the main elements lumen (Rodrigues & Gómez, 1999). that constitute the composition of these struc- The penis is highly muscular with the wall tures in O. atlanticus in order to know their thickening towards the atrium. About a quarter nature and to compare with other inorganic way from the atrium there is a conspicuous deposits subsequently found in other stylom- sheath of muscular-connective tissue which dis- matophorans. tally fuses with the penis wall. At the proximal end of the penis is a penial caecum (Fig. 1a); this is folded, and the wall slender and less MATERIALS AND METHODS muscular than the rest of penis. The columnar Adult specimens of O. atlanticus were captured in the epithelium is surrounded by connective tissue field in July, at the beginning of the mating season of with muscular fibres on it. A thin muscular this species. Copulating pairs, as well as non-copula- layer, thicker in the distal penis, is external to ting specimens were collected and immediately frozen this. The vas deferens is a long slender duct with by immersion in liquid nitrogen. Alternatively, speci- three or four longitudinal folds. It is replaced mens were kept alive in the laboratory until process- distally by the epiphallus, a slightly enlarged ing. In both cases, the soft parts were withdrawn from duct with five or six longitudinal folds (Fig. 1b). the shell and dissected under a stereomicroscope. There are many granular cells in the connec- Isolated reproductive distal organs, vas deferens, epi- phallus, and penis with penial caecum (ϭflagellum tive wall of these organs. They have their cyto- of several authors working with zonitids) were dehy- plasm full of granules, spheroidal in shape (Figs drated with ethanol (70%, 96%, 100%). In copulating 1c,d,e). They vary in size, but the majority of specimens the whole copulatory organs, from the them measure from 0.5 ␮m to 2.5 ␮m in diam- atrium to the bursa copulatrix, were removed and eter; the largest can measure up to 4 ␮m in treated together. Fixatives or buffers were not used diameter. Transverse sections of the penial for the preparation of this material in order to avoid caecum show that this cell type represents the potential contamination of tissues with exogenous almost all the connective element of its wall chemical elements. To perform scanning electron (Fig. 1a). It can also be the main cell type in the microscopy (SEM) observations, the isolated organs were immersed once more in liquid nitrogen and a connective portion of the epiphalic folds (Figs. transverse cryofracture was made. Afterwards, these 1b,d). They are also present, but more scattered, organs were returned to 100% ethanol. They were in the distal portion of the vas deferens and in then treated in a critical point dryer (Balzers, the proximal penis, beyond the penial caecum. CPD020), coated with gold in a Bal-tec SCD 004 These granular cells can also be observed in the Sputter coater, and observed in a JEOL SEM 6400. thin outer muscular layer that surrounds all these The X-ray microanalyses were performed with a male distal organs (Fig. 1b) as well as in the energy-dispersive spectrometer (EDX) associated penial sheath. Several morula-like cell bodies of with the SEM microscope. The SEM was operated this cell type can be seen quite distinctly pro- at 20 kV. Granules or spermatophore tunicle were probed centrally with the beam operating as a station- truding on the external wall of the ducts (Fig. ary spot. X-ray spectra were obtained with a Link 2a,b), unless they are stretched; the roundness microanalyser. The acquisition time was the same for of the granules they contain is noticeable under all the analysis (100 seconds real time), thus allowing the cell membrane (Figs. 1c, 2b). The X-ray a direct comparison of the amount of metals between microanalysis demonstrates that the main ele- the different spectra. ments of these spherules are calcium and phos- phorus (Figs. 1A ϩ 3B). No other elements were detected. Elements with atomic number below RESULTS that of sodium, such as carbon, hydrogen, nitro- gen, and oxygen are not detected by the used The distal portion of the male genital system of techniques. O. atlanticus consists of vas deferens, epiphal- A transverse section made across the everted lus, and penis, with a penial caecum originating penis in copulating specimens shows the epi- at the subterminal insertion of the epiphallus on phallus being enclosed by the penis (Figs. 2c,d). the penis. In the female distal part there is a The wall of the latter is turned over with the bursa copulatrix with its duct, a free oviduct, dense microvilli of its epithelium placed on the and the vagina with a perivaginal gland. A short external surface (Fig. 2c) while he outer thin common atrium opens on the body wall. During muscular layer of penis and epiphallus are face 07 197-204 Gomez 31/3/0 10:59 am Page 199 CALCIUM PHOSPHATE GRANULES IN OXYCHILUS ATLANTICUS 199 to face. Several granular cells of both outer Frequently, it is possible to find a spermato- muscular layers are ripped and the spherules phore in specimens killed during copula. It can they contain released into the quite narrow be found inside the proximal portion of the space that exists between penis and epiphallus epiphallus, before having been transferred, or (Fig.