J. Moll. Stud. (1991), 57: 309-322. © The Malacological Society of London 1991 COMPARATIVE SPERMATOLOGY OF FOUR SYMPATRIC SPECIES OF SIPHONARIA (PULMONATA: BASOMMATOPHORA) A.N. HODGSON, R.T.F. BERNARD and D.S. LINDLEY Department of Zoology & Entomology, Rhodes University, Grahamstown 6140, South Africa (Received 25 April 1990, accepted 1 September 1990) Downloaded from https://academic.oup.com/mollus/article/57/3/309/976378 by guest on 27 September 2021 ABSTRACT although these have been largely restricted to prosobranchs (see Healy 1988c for review). Far The fine structure of the modified sperm and sper- less is known about sperm structure and sper- matogenesis of four sympatric species of Siphonaria matogenesis of the euthyneuran gastropods is described. The morphology of the sperm of all species is very similar. The head, which is about 6 um (Healy & Jamieson, 1989) and within the Pul- long, is composed of a nucleus with fibrous chromatin monata there are only a few comparative studies capped by an acrosome (about 1 urn long) comprised on closely related species (Healy, 1983b; Sumi- of an acrosomal pedestal and apical vesicle. The mid- kawa & Funakoshi, 1984; Healy & Jamieson, piece has a mitochondrial derivative which surrounds 1989). It has been suggested that within the pul- a single glycogen helix, posterior to which is a glycogen monates, the order Basommatophora may not piece. Although differences between each species constitute a validgroup (Hubendick, 1978; Healy exist, the value of sperm morphology for purposes of 1983b), and Haszprunar (1988) in a review of the taxonomy in this genus is questioned. Comparison origin and evolution of the gastropods suggests with other basommatophorans however suggests that that the relationship between, and taxonomy of, sperm morphology may be of value at a higher taxo- nomic level. The morphological changes that occur the various groups of pulmonate needs to be during spermatogenesis are similar to those described reconsidered taking into account characters such for other molluscs with modified sperm, except that as spermatozoon morphology. If spermatozoon during early spermiogenesis the Golgi body and morphology is to be of vatue in examining phylo- smooth endoplasmic reticulum become highly devel- genetic relationships between pulmonates, oped. This proliferation of the SER and Golgi occurs further comparative investigations are required, at the same time as elongation of the spermatid. particularly between closely related taxa. Throughout spermatogenesis, the germ cells are closely associated with a somatic cell which, because The intertidal zone of the coast and estuaries of structural similarities with the somatic cell of mam- of the eastern Cape of South Africa is inhabited malian seminiferous epithelium, has been termed a by four species of Siphonaria which are members Sertoli cell. After the spermatids have been released of the pulmonate order Basommatophora. The from the Sertoli cells of the testis, maturation con- spermatozoa of these species were briefly tinues in the hermaphrodite duct where the acrosome described in an abstract by Hartley, de Villiers reaches its final size and glycogen accumulates in the & Hodgson (1985). All measurements for this glycogen compartment of the mid-piece. paper were made by scanning electron micro- scopy, and subsequent analysis (by A.N.H.) revealed that these measurements were inac- INTRODUCTION curate. In this paper we correct this, describe and compare spermatogenesis and provide more A number of recent studies have shown the detail on the structure of the spermatozoon of value of spermatozoon morphology in assessing the four sympatric species of Siphonaria. In the phylogenetic and taxonomic relationships of addition, the structure of the sperm is compared molluscs (Healy, 1982; 1983a, b; 1988a, b, c; with that of other pulmonates and similarities Healy & Willan, 1984; Healy & Jamieson, used to draw conclusions about possible phylo- 1989; Hodgson & Bernard, 1986a, b; 1988; genetic affinities. Hodgson, Baxter, Sturrock & Bernard, 1988; Hodgson, Bernard & van der Horst, 1990; MATERIALS AND METHODS Hodgson, Heller & Bernard, 1990). Such studies have been particularly useful in examining Specimens were collected in January and February relationships within the class Gastropoda, 1989 from the rocky shores of the Eastern Cape 310 A.N. HODGSON, R.T.F. BERNARD & D.S. LINDLEY (34°15'S, 18°30'E) of South Africa and transported 5 urn diameter). The nucleus contains one or back to the laboratory where portions of the ovotestes two nucleoli (about 0.7 urn diameter) and small and hermaphrodite ducts were prepared for either clumps of electron-dense chromatin which often light or transmission electron microscopy. have a peripheral distribution (Fig. lc). The cytoplasm possesses only a few small oval mito- Light microscopy chondria (about 0.4 \un x 0.3 urn) and a small Spermatozoa were obtained by dissecting the her- amount of smooth endoplasmic reticulum. maphrodite duct from the animal and placing it in a The oval nucleus of spermatocytes is smaller drop of sea water on a microscope slide. The duct was (about 4 \im x 2 \im) than that of the sper- teased with a mounted needle and a smear made matogonium and the electron-dense chromatin of the released spermatozoa. The total length, head is organized as a patchwork. A nucleolus is Downloaded from https://academic.oup.com/mollus/article/57/3/309/976378 by guest on 27 September 2021 length and wavelength of glycogen compartment present in early spermatocytes and this often has which spirals as a helix along part of the mid-piece (see a peripheral location (Fig. Id). The cytoplasm results) were measured for 20 spermatozoa (xlOOO magnification) using a Nikon Filar micrometer contains a number of large mitochondria (about eyepiece. This procedure was repeated for five indi- 0.7 urn x 0.4 |im), a small Golgi body (about 5 viduals of each species. An analysis of variance was cisternae) as well as a little smooth endoplasmic then employed to determine whether there was any reticulum (Fig. Id). significant difference in head length and helix wave- The nucleus of early spermatids contains length within and between individuals of a species. chromatin which is scattered throughout, and Student's /-tests were also performed to determine within the cytoplasm there have been increases whether there were any significant differences in the number of mitochondria (which have between species. prominent lamellar cristae); the amount of smooth endoplasmic reticulum; and the size of Transmission electron microscopy the Golgi body (Fig. 2a, b) which can comprise Portions of the ovotestes and hermaphrodite ducts up to 19 cisternae. Small (0.1 nm diameter) were dissected from the limpets and fixed in 2.5% membrane-bound vesicles, presumed to be pro- glutaraldehyde in filtered sea water and 0.1 M sodium acrosomal vesicles, are often seen in close cacodylate buffer (pH7.4 and isosmotic with the association with the Golgi body (Fig. 2b). As tissues) at 4°C for 12 h. After washing in 0.1 M cac- maturation proceeds the nucleus becomes odylate buffer the tissues were postfixed in 1 % osmium spherical (about 3 \xm), the chromatin begins to tetroxide in sodium cacodylate buffer for 90min, condense, becoming granular in appearance, dehydrated in a graded ethanol series and embedded and the nuclear membranes thicken, firstly at in an Araldite/Agar 100 resin mixture via propylene the presumptive posterior (Fig. 2b) and later at oxide. Semi-thin sections were stained in toluidene blue and thin sections (silver/gold interference) the presumptive anterior (Fig. 2c). Within the stained in either 5% aqueous uranyl acetate or 2% cytoplasm the mitochondria accumulate at the methanolic uranyl acetate, followed by lead citrate. presumptive posterior of the cell (Fig. 2b, c, d), Some sections were stained for gJycogen using the and there is further proliferation of the smooth periodic acid-sodium chlorite-uranyl acetate tech- endoplasmic reticulum which is now positioned nique (Vye & Hschman, 1971). Sections were exam- anteriorly around the nucleus (Fig. 2b, c). ined with a Jeol 100 CXII electron microscope. As development progresses to a mid-sper- matid (Fig. 2d), the nucleus becomes oval (about 3.7 |im x 1.5 |im), invaginated posteriorly and the chromatin continues to con- RESULTS dense (Fig. 2d). The centrioles, from which the tail has already begun to develop, occupy the Spermatogenesis posterior invagination of the nucleus. The mito- Spermatogenesis is similar in all four species and chondria accumulate around the developing tail a single description follows. where they fuse to form a mitochondrial deriva- Light microscopy reveals that most stages of tive (Fig. 3a, c). The mitochondrial derivative spermatogenesis and some stages of oogenesis encircles a small region of the cytoplasm around can be observed in the ovo-testis (Fig. la, b). the axoneme, which eventually becomes the During maturation the developing spermatozoa glycogen compartment. During this stage the are clearly associated with large somatic cells axoneme undergoes considerable elongation (Fig. la, b) which we presume to be Sertoli cells. and as it does so, cytoplasm and the developing Spennatogonia are sited close to the wall of mitochondria derivative migrate with it (Fig. each germinal follicle and each spermatogonium 3b). Cytoplasmic migration appears to halt at is characterized by a large found nucleus (about the junction of the mid-piece and the glycogen SPERM OF SIPHONARIA 311 Downloaded from https://academic.oup.com/mollus/article/57/3/309/976378 by guest on 27 September 2021 Figures la & b. Siphonaria capensis. Light microscope sections through the gonad showing some stages of spermatogenesis (arrowed). Abbreviations: O, developing egg; S, Sertoli cell; Sts, spermatids. Scale bars = 0.1 mm. Figure lc. Siphonaria aspera. A spermatogonium adjacent to the wall (W) of the germinal follicle from the gonad of S. aspera. Scale bar = 2 um. Figure Id. Siphonaria aspera. A spermatocyte from the gonad. Scale bar = 1 um. Abbreviations: G, Golgi body; M, mitochondrion; N, nucleus; No, nucleolus; SER, smooth endoplasmic reticulum.