AN ULTRASTRUCTURAL STUDY OF OOGENESIS IN A PLANKTONIC AND A DIRECT-DEVELOPING SPECIES OF SIPHONARIA (GASTROPODA: PULMONATA) PURBA PAL AND ALAN N. HODGSON Department of Zoology and Entomology, Rhodes University, Grahamstown, 6140, South Africa (Received 7 January 2002; accepted 27 March 2002) ABSTRACT Oogenesis and vitellogenesis were compared at an ultrastructural level in Siphonaria capensis (a plank- Downloaded from https://academic.oup.com/mollus/article-abstract/68/4/337/1004678 by guest on 07 September 2019 tonic developer) and S. serrata (a direct developer). Except for some months in winter, most stages of oogenesis were observed during the year within a gonad, although oogenesis was asynchronous between the gonad acini. Previtellogenic oocytes, which contained few organelles, were surrounded by follicle cells. During vitellogenesis three types of storage products were accumulated in the ooplasm: yolk, lipid and glycogen. In S. capensis yolk formation begun before lipid synthesis and the yolk was produced autosynthetically. By contrast in S. serrata lipid was deposited before yolk synthesis. Morpho- logical evidence (production of yolk by Golgi bodies and rough endoplasmic reticulum; endocytotic pits along the oolemma) was found for yolk formation by both auto and heterosynthesis. In both species as the oocytes grew the follicle cells became hypertrophic and then gradually withdrew from the oocytes. Results from this study add further support to the suggestion that siphonariid limpets had a marine ancestry. INTRODUCTION Siphonaria, there have been no descriptions of egg formation (oogenesis and vitellogenesis; Hodgson, 1999). While the life Siphonariid limpets are very common pulmonates in the inter- history strategy or developmental mode is constrained by ances- tidal regions of warm temperate to tropical rocky shores, try (as has been shown in littorinids, Reid, 1990) some studies especially in the southern hemisphere (Hodgson, 1999). As have shown that there is a close correlation between the type of basommatophorans they are considered primitive pulmonates oogenesis and life history pattern (Eckelbarger, 1994). Oogene- (Hubendick, 1978), but their ancestry is uncertain. The pres- sis and associated modes of vitellogenesis also set interspecific ence of planktonic development in many species of Siphonaria differences during egg development and for this reason Siphon- and their lack of tentacles, has led some authors to argue for a aria presents an opportunity to compare the process of oogen- marine ancestry (Hyman, 1967; Purchon, 1979). By contrast esis in closely related species from the same habitat, but with others have suggested that siphonariids have a terrestrial origin different modes of development. The aim of this work was to and they re-invaded marine environments (e.g. Hubendick, compare oogenesis and vitellogenesis in species of Siphonaria 1947; Borland, 1950; Yonge, 1952). The more recent work with planktonic (S. capensis Quoy & Gaimard, 1833) and direct of Chambers, McQuaid & Kirby (1996, 1998) did not clarify development (S. serrata Fischer, 1807), testing the hypothesis matters. Their results from analysis of total proteins suggested that different modes of development (with the production of that planktonic development and marine ancestry (Chambers eggs of different sizes) would involve differences in oogenesis. et al., 1996) was primitive, whereas the later information from DNA fingerprinting (Chambers et al., 1998) indicated the oppos- ite, i.e. siphonariids are descended from terrestrial ancestors MATERIAL AND METHODS with direct development. Specimens of Siphonaria capensis and S. serrata were collected Like all pulmonates, siphonariids are hermaphrodites with from intertidal rocks at Kenton-on-Sea (33Њ42Ј S, 26Њ41Ј E) in internal fertilization. They lay fertilized eggs on rocks, the eggs the Eastern Cape, South Africa. To ensure that all stages of being protected by capsules, which are embedded in a jelly oogenesis were obtained, three specimens of each species were matrix (Hodgson, 1999, for review). Although these limpets collected seasonally. Samples were collected twice in spring have been studied extensively, many aspects of their repro- (September and October, 1999) and summer (December, ductive biology are still unknown (Hodgson, 1999). Two main 1999, and February, 2000), once in autumn (May, 2000) and larval developmental patterns, planktonic and direct, have been winter (July, 2000). Animals were transported back to the labor- recorded for the genus Siphonaria (Chambers & McQuaid, atory where the gonad was removed. Small portions of the 1994a,b). Planktonic developers lay large numbers of small eggs, gonad were fixed for approximately 12 h in cold 2.5% which hatch after 4–5 days as veliger larvae, whereas direct glutaraldehyde in 0.1 M sodium cacodylate buffer and filtered developers lay smaller numbers of larger eggs from which sea-water (pH 7.2). After fixation, tissues were washed in 0.2 M crawling juveniles emerge after 3–4 weeks (Chambers, 1994; sodium cacodylate buffer (pH 7.0) and postfixed in 1% OsO Chambers & McQuaid, 1994a,b; Hodgson, 1999). Species with 4 in sodium cacodylate buffer for 90 min at room temperature. both forms of development can be found in sympatry on South After rinsing the tissues in two changes of buffer they were dehy- African shores (Allanson, 1958; Chambers, 1994). drated in ascending concentrations of ethanol to 100%. Tissues The occurrence of different life history strategies in siphon- were infiltrated (via propylene oxide) and embedded in an ariids has been explained in terms of adaptation and ancestry Araldite/Taab mixture (Cross, 1989). Both semi-thin and (Chambers, 1994). Although the type of development and size ultra-thin sections were cut using glass knives on a RMC MT7 of egg or egg capsule are well known for numerous species of ultramicrotome. Semi-thin sections, stained in 1% toluidene Correspondence: P. Pal; e-mail: [email protected] blue dissolved in 2.5% sodium carbonate, were observed and J. Moll. Stud. (2002) 68: 337–344 © The Malacological Society of London 2002 P. PAL & A. N. HODGSON photographed with a light microscope. Ultrathin sections were of which are closely associated with lipid (Fig. 2E). Yolk granules stained in 5% aqueous uranyl acetate (30 min) and Reynold’s begin to appear once lipid formation is underway (see insert to lead citrate (5 min), and the grids were viewed with JEOL 1210 Fig. 2F). transmission electron microscope at 100 kV. As vitellogenesis proceeds in both species, the proteosyn- thetic organelles increase in number, the mitochondria pro- RESULTS liferate and elongate (especially in S. serrata; Fig. 2F). There is an increase in the number and size of the yolk granules, which The gonad of siphonariids, including the species studied here, gradually fill the ooplasm from the centre outwards, eventually is composed of numerous closely grouped acini in which both reaching a size of about 2–5 ␮m in diameter (Figs 2C, F, 3A, eggs and sperm develop (Marcus & Marcus, 1960; Berry, 1977; F). In addition, they also show changes in their structure. In Hodgson, Bernard & Lindley, 1991; Luchtel, Martin, Deyrup- both S. capensis and S. serrata the contents of the electron-dense Olsen & Boer, 1997). Except for winter months (June/July), yolk granules begin to differentiate into a crystalline core sur- most stages of oogenesis could be found all year round within a rounded by an electron-lucent cortex (Fig. 3A, B, F). Downloaded from https://academic.oup.com/mollus/article-abstract/68/4/337/1004678 by guest on 07 September 2019 gonad, but oogenesis was asynchronous between acini (Fig.1A, In the mid- to late vitellogenic oocytes of S. serrata endocytotic B). Early oocytes lie next to the wall of the acinus and as they pits form along the oolemma (Fig. 3D). From this endocytotic grow and mature they gradually fill its lumen (Fig. 1A, B). activity vesicles are produced which fuse to produce yolk gran- The wall of each acinus is about 0.8–1.0 ␮m thick and consists ules (about 1.5–2 ␮m diameter) in the cortical region of the of a layer of thin cells, which often contain pigment granules, a oocyte (Fig. 3E, F). These granules, which reach a maximum small amount of smooth muscle and a band of fibrous connect- size of 2 ␮m in diameter, have a very electron lucent cortex with ive tissue (Fig. 1F, G, H). a granular core (Fig. 3E). They are restricted to the cortical region of the egg and may represent a second type of yolk Previtellogenic oocytes granule. Also present in the cortical region are arrays of smooth ER, which are closely associated with lipid droplets (Fig. 3C). In The structure of the previtellogenic oocytes in both species is both species, glycogen granules appear in the ooplasm at this very similar (Fig.1C, D). The smallest oocytes observed were stage (not illustrated). The yolk granules reach a size of approx- about 15 ϫ 12 ␮m in size. Early previtellogenic oocytes possess a imately 4–5 ␮m in S. capensis and 2–3 ␮m in S. serrata. large, round nucleus (about 8–10 ␮m diameter) with scattered The oocytes, which reach a maximum size of 70–100 ␮m in heterochromatin and a prominent nucleolus (Fig. 1C). The S. capensis and 90–150 ␮m in S. serrata, are surrounded by ooplasm contains a few round mitochondria (about 0.5 ␮m follicle cells throughout oogenesis. During vitellogenesis, the diameter) with prominent cristae and small amounts of endo- follicle cells show an increase in the number of proteosynthetic plasmic reticulum (Fig. 1D). As the oocytes grow, the nucleus organelles especially arrays of rough endoplasmic reticulum, develops more than one nucleolus (one large amphinucleolus Golgi bodies and some lysosomes (Fig. 4A–D). In addition, the about 9 ϫ 8␮m and a small eunucleolus about 4 ␮m diameter) follicle cells accumulate electron-dense granules, which are pre- (Fig. 1E). sumed to be glycogen (Fig. 4G). Follicle cells seem to contain Previtellogenic oocytes are separated from the wall of the microtubules at all stages of oogenesis (Fig.