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 (3342 S, 2641 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 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 8m 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. 4E). By late vitello- acinus by follicle cells (Fig. 1C) that completely surround each genesis, the follicle cells are very squamous and are becoming oocyte. Each follicle cell has a large nucleus (about 7 3 m) detached from the oocytes creating an intercellular gap between with scattered heterochromatin and a single nucleolus (Figs 4A, the follicle cell and oocyte (insert to Fig. 4E, F). The oolemma of 1C). The cytoplasm contains scattered rough endoplasmic the oocytes then begins to form simple microvilli with glycocalyx reticulum, Golgi bodies and tubular mitochondria with promin- (Fig. 4D, F). ent lamellar cristae (Fig. 4B, C). The Golgi bodies produce small vesicles, which are membrane-bound with electron-dense DISCUSSION material inside (Fig. 4C). Some dense bodies are also found in the cytoplasm (Fig. 4B, C). Cell junctions connect adjacent The general structure of the gonad of Siphonaria capensis and follicle cells as well as the oocytes to the follicle cells (Fig. 4F, H). S. serrata does not differ from that of other pulmonates (Jong- Follicle cells lie adjacent to the connective tissue wall of the Brink, Boer & Joosse, 1983; Luchtel et al., 1997). It consists acinus, but do not appear to be attached to it by cell junctions of numerous acini in which both male and female gametes (Fig. 1G). develop. The oocytes develop peripherally next to the wall of the acinus and as they mature they gradually fill its lumen. In Vitellogenic oocytes siphonariid limpets the acinar wall is composed of connective tissue, muscles and squamous cells with pigment granules, As vitellogenesis commences the nucleus of the oocytes of both which is in agreement to what has previously been described in species increases in size to approximately 30 40m and other pulmonates (Sabelli & Sabelli-Scanabissi, 1982; Luchtel becomes lobular in shape (Fig. 2A). There is also an increase et al., 1997). in the number of proteosynthetic organelles (Fig. 2A, B). In The formation of yolk by autosynthesis is widespread in mol- S. capensis small, spherical mitochondria accumulate close to luscs (Wourms, 1987; Eckelbarger & Davis, 1996) and it has the nuclear membrane along with arrays of rough endoplasmic been suggested that this is the primary yolk-forming means in reticulum, some Golgi bodies and nuage-like material (Fig. this phylum (Jong-Brink et al., 1983; Medina, Garcia, Moreno 2A–D). Vitellogenesis starts perinuclearly with the appearance & Lopez-Campos, 1986). Nevertheless, the incorporation of of small electron-dense vesicles (about 0.8–1.0 m diameter), exogenous yolk precursors by endocytosis is also well document- which we presume to be nascent yolk granules, forming close to ed (Bottke 1973, 1986; Hill & Bowen, 1976; Selman & Wallace, the rough endoplasmic reticulum (Fig. 2B–D). Other small ves- 1978; West, 1981; Bottke, Sinha & Keil, 1982; Khan & Saleuddin, icles, which appear to be produced by Golgi bodies, fuse with 1983; Eckelbarger & Blades-Eckelbarger, 1989; Eckelbarger & the nascent yolk granules (Fig. 2G, H). Once yolk granule Young, 1997; Hodgson & Ecklebarger, 2000). Results of the formation is underway, lipid droplets about 1 m in diameter present study indicate that the planktonic developer, S. capensis begin to accumulate in the ooplasm (Fig. 2C). produces membrane-bound yolk granules by autosynthesis In S. serrata, early vitellogensis is characterized by prolifera- only, which was manifested by the increase in the number of tion of smooth endoplasmic reticulum and mitochondria, both proteosynthetic organelles (e.g. rough endoplasmic reticulum

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1 2 3 4 5 6 7 8 9 10 1 2 3 Downloaded from https://academic.oup.com/mollus/article-abstract/68/4/337/1004678 by guest on 07 September 2019 4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 2 3 4 5 6 7 8 9 40 1 2 3 4 5 6 7 8 9 50 1 2 3 4 5 6 7 8 9 60 1 2 Figure 1. A, B. Light micrographs of toluidene blue stained sections showing different stages of oogenesis in the gonad of Siphonaria capensis. 3 C, D. Previtellogenic oocytes of S. serrata and S. capensis, respectively. The ooplasm contains a spherical nucleus (n), a few small mitochondria (m), rough endoplasmic reticulum (rer) and in S. serrata a few lipid droplets (l). The oocytes are surrounded by follicle cells (f), containing spherical nucleus (n) with 4 a nucleolus (arrowhead). E. Previtellogenic oocyte of S. capensis in which the nucleus (n) contains a large amphinucleolus (an) and a small eunucleolus (en). 5 Note the band of mitochondria (m) around the nuclear membrane (arrowhead). F–H. Electron micrographs of the wall of gonad acini. The wall (w) consists 6 of a layer of thin cells containing pigment granules (pg) with an irregularly-shaped nucleus (n), connective tissue (ct) and muscle (mu). Scale bars: A, B 7 35 m; C 2 m; D 500 nm; E 1 m; F 2 m; G 1 m; H 200 nm. Abbreviations: ev, early vitellogenic oocyte; h, heterochromatin; mo, mature 8 oocyte; p, previtellogenicoocyte; vo, vitellogenic oocyte.

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Figure 2. A. Early vitellogenic oocyte of Siphonaria capensis with rough endoplasmic reticulum (arrowheads), a few yolk granules (yg). B. Early vitellogenic oocyte of S. capensis showing dense bodies (db), which are probably nascent yolk granules and a band of mitochondria (m) close to the nucleus (n). C. Vitellogenic oocyte of S. capensis in which yolk synthesis is underway and lipid droplets (l) have begun to appear in the ooplasm. Extensive arrays of rough endoplasmic reticulum (arrowhead) are seen in the perinuclear cytoplasm. D. Early vitellogenic oocyte of S. capensis showing rough endoplasmic reticulum (rer), a nascent yolk granule (yg) and nuage (ng). E. Early vitellogenic oocyte of S. serrata with lipid droplets (l) and smooth endoplasmic reticulum (ser). F. Perinuclear ooplasm of a vitellogenic oocyte of S. serrata showing lipid droplets (l), elongated mitochondria (m), lobular nucleus (n) and yolk granules (yg). Insert: nuage like material (ng) in S. serrata. G. Early vitellogenic oocytes of S. capensis showing Golgi body (g) producing small vesicles (v). H. Vesicles (v) fusing to form mature yolk granule (yg) in early vitellogenic oocyte of S. capensis. Scale bars: A–C 1 m; D 200nm; E 1 m; F 1m; insert 1m; G 200 nm; H 200 nm. Abbreviations: f, follicle cell; rer, rough endoplasmic reticulum.

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1 2 3 4 5 6 7 8 9 10 1 2 3 Downloaded from https://academic.oup.com/mollus/article-abstract/68/4/337/1004678 by guest on 07 September 2019 4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 2 3 4 5 6 7 8 9 40 1 2 3 4 5 6 7 8 9 50 1 2 3 4 5 6 7 8 9 60 1 2 3 Figure 3. A. Late vitellogenic oocyte of Siphonaria capensis showing lipid (l) and yolk granules (yg) with bipartite structure. B. Higher magnification of yolk gran- 4 ules from S. capensis shows the crystalline core (c) and electron lucent cortex (co). C. Late vitellogenic oocyte of S. serrata showing lipid droplet (l) in close con- 5 tact with smooth endoplasmic reticulum (ser) in the cortical ooplasm. D. Endocytotic pit (p) forming along the oolemma (ol) in a late vitellogenic oocytes of S. 6 serrata. E. Vesicles fusing (arrowed) to form yolk granule (yg) in the cortical ooplasm of S. serrata oocytes. F. Late vitellogenic oocyte of S. serrata showing 7 the prevalence of lipid (l) in the ooplasm. Scale bars: A 1 m; B 100 nm; C 200 nm; D 200 nm; E 200 nm; F 2 m. Abbreviations: f, follicle cell; is, 8 intercellular space; mv, microvilli; m, mitochondria; w, wall of acinus.

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Figure 4. A. Follicle cell (f) between two early vitellogenic oocytes of Siphonaria capensis. B. Follicle cell (f) surrounding an early vitellogenic oocyte of S. serrata. C. Follicle cell cytoplasm showing Golgi bodies (g) producing small vesicles (v). D. Two mid-vitellogenic oocytes (mvo) and follicle cells (f) with extensive arrays of rough endoplasmic reticulum (rer). E. Follicle cell (f) shows presence of microtubules (mt). Insert: squamous follicle cells (f) between two mid-vitellogenic oocytes (mvo) of S. capensis. F. Micrograph showing cell junction (arrowhead) between two follicle cells (f). Microvilli (mv) of an oocyte of S. serrata can also be seen. G. Follicle cell cytoplasm showing glycogen (gl) in S. capensis. H. Cell junction (arrowhead) between a follicle cell (f) and an oocyte (o) of S. serrata. Scale bars: A 2 m; B 500 nm; C 200 nm; D 1 m; E 500 nm; insert 500 nm; F 500 nm; G 200 nm; H 200 nm; I 500 nm. Abbreviations: w, acinar wall; db, dense bodies; ly, lysosome; m, mitochondria; mv, microvilli; yg, yolk granule.

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1 and Golgi bodies). The involvement of RER and Golgi bodies in Oogenesis, life history and the evolution of siphonariids 2 yolk production is well documented (Bedford, 1966; Taylor & 3 Anderson, 1969; Jong-Brink, Wit, Kraal, Boer, 1976; West, 1981; Although both S. capensis and S. serrata have eggs, which contain 4 Kress, 1986; Wourms, 1987; Eckelbarger & Davis, 1996). By yolk, lipid and glycogen, the oocytes of S. serrata (the direct 5 contrast, in S. serrata yolk appears to be formed both auto- and developer) are larger and clearly must contain a greater quan- 6 heterosynthetically. Like S. capensis, the majority of the yolk tity of storage product. This difference in size of the eggs of 7 granules are formed autosynthetically at the beginning of vitello- planktonic and direct developers confirms earlier observations 8 genesis in the perinuclear cytoplasm. As vitellogenesis proceeds on siphonariids (Hodgson, 1999). Our qualitative observations, 9 some yolk develops in the cortical region of the oocyte. These however, suggest that the type and proportion of the storage 10 yolk granules (which may represent a second type) appear to be product is not the same in these species, with oocytes of S. serrata 1 formed by heterosynthesis with coated pits developing along containing more lipid. Presumably, these additional energy 2 the oolemma. These, in turn, form vesicles, which fuse to form reserves are required to sustain intracapsular development in 3 the yolk granules. Yolk formation from endocytotic activity this species. Whether the embryos of S. serrata obtain any nutri- Downloaded from https://academic.oup.com/mollus/article-abstract/68/4/337/1004678 by guest on 07 September 2019 4 during mid- to late vitellogenesis has been observed in other ents from capsule fluids as has been shown for Littorina (Moran, 5 molluscs (e.g. Spurilla and Bathynerita; Eckelbarger & Blades- 1999), has still be determined. Similar differences in the quan- 6 Eckelbarger, 1989; Eckelbarger & Young, 1997). The presence tity of storage products within oocytes have also been observed 7 of these pits in the cortical ooplasm indicates possible uptake of between oviparous (egg-laying) and ovoviviparous (brooding) 8 large molecular weight extra-ovarian substances. The source freshwater gastropods (Hodgson et al., in press). 9 and nature of these extra-oocytic substances needs to be estab- We suggest that the provisioning S. serrata eggs with addi- 20 lished, but it is possible the follicle cells (which are proteo tional energy reserves is facilitated by the use of more than one 1 synthetically active) may be their source. In the polychaete mode of vitellogenesis. Unlike S. capensis where we found 2 worm Capitella jonesi the follicle cells were suggested to be most evidence for autosynthesis, yolk is formed by both auto- and 3 probable site for extra-oocytic substances (Eckelbarger & Grassle, heterosynthesis (mixed synthesis). Autosynthesis is considered 4 1982). In siphonariids apart from RER and Golgi bodies no to be the primitive mode of vitellogenesis in monotelic breeders 5 other organelle was involved in producing yolk autosynthetical- (Eckelbarger 1994). The fact that S. capensis has this mode of 6 ly unlike in Planorbis corneus where mitochondria participate in synthesis only, supports the hypothesis that planktonic develop- 7 producing protein yolk (Favard & Carasso, 1958). ment is primitive and that siphonariids, therefore, could have 8 The yolk granules in both species have a bipartite structure, a had a marine ancestry. However, further studies should be under- 9 common feature of mollusc yolk, as well that of other inverte- taken to ascertain whether the vitellogenic modes described for 30 brates (Nørrevang, 1968; Wourms, 1987; Eckelbarger & Blades- S. capensis and S. serrata are typical of planktonic and direct 1 Eckelbarger, 1989). Such yolk granules or yolk platelets typically developers, respectively. 2 consist of a protein yolk core surrounded by other carbohydrate 3 moieties (Gérin, 1976; Wourms, 1987). ACKNOWLEDGEMENTS 4 Apart from membrane-bound yolk granules, lipid accu- We would like to thank the staff of the electron microscopy 5 mulates in the ooplasm of both species of Siphonaria during unit for technical and photographic services. This project was 6 early vitellogenesis. We were unable to determine how the lipid funded by the National Research Foundation, South Africa and 7 might be formed, but in S. serrata lipid droplets developed in the Malacological Society of London (Centenary Grant, 2000). 8 close association with mitochondria and smooth endoplasmic 9 reticulum. Kessel (1982) suggested that in the patellogastropod 40 Acmaea, annulate lamellae and mitochondria play a role in lipid REFERENCES 1 formation. To date, we have not observed annulate lamellae in ALLANSON, B.R. 1958. Some aspects of the ecology of the molluscan 2 the oocytes of Siphonaria. genus Siphonaria in South Africa. 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