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Neritid egg capsules: are they all that different? KOH SIANG TAN & SERINA S. C. LEE

Steenstrupia

Tan, K. S. & S. S. C. Lee. Neritid egg capsules: are they all that different? – Steenstrupia 30 (2): 115–125. Copenhagen, Denmark. April 2009. ISSN 0375-2909. Egg capsules deposited by Nerita and other related genera of the neritopsine gastropod family Neritidae are a familiar sight on tropical shores. The small, white to grey flattened discs are laid over rocks, stones, wood, mollusc shells and other hard surfaces in the intertidal zone. Differences in morphology were at first thought to be slight. All capsules comprise a shallow, thin-walled tambour attached to the substratum, covered by a thick-walled, dome-shaped or flat cap-like cover, enclosing the embryos within. The lower tambour is mostly a thin organic membrane that adheres to the substratum, but also has a distinct raised, thickened rim. Fine grooves and ridges are present on the inside surface of the rim. The upper cap or cover is a more robust structure that fits over the tambour, with a corresponding region of grooves and ridges along its rim. Cover and tambour are firmly attached to each other along their rims during embryonic development. They separate partially or completely to release tens of swimming veliger larvae. The external surface of the cover may be embedded with calcareous spherulites derived from a special crystal sac within the female, or with foreign material presumably obtained from the environment via the digestive system. Detailed observations show that each of six species of Nerita in Singapore has a unique arrangement and size of calcareous spherulites embedded in the capsule surface. Spherulites of some species also show distinct refractive properties when viewed under polarized light. The egg capsules of N. planospira lack spherulites altogether, and the surface is instead furnished with fine outgrowths of the organic matrix. The overall arrangement and particle sizes of sand and silt also differ in Clithon oualaniensis and Dostia violacea. These dissimilarities are sufficient to distinguish the majority of species. Keywords: Egg capsule morphology, Neritidae, Nerita, Clithon, Dostia, spherulite K. S. Tan & S. S. C. Lee: Tropical Marine Science Institute, National University of Singapore, 18 Kent Ridge Road, Singapore 119227. E-mail: [email protected]

INTRODUCTION Lens-shaped egg capsules laid by gastropods in the genus Nerita and other related neritopsine genera are conspicuous and often abundant on hard intertidal shorelines across Southeast Asia, reflecting the ubiquitousness and success of this enigmatic group of gastropods on tropical shores. Previous work, notably by Andrews (1933, 1935, 1937), Lamy (1928), Risbec (1932) and later by Knudsen (1992, 1997) pointed to possible differences in egg capsule structure between different species, as well as between genera. The egg capsules of intertidal neritid species differ from most other seashore gastropods in having their external surface reinforced with minerals, possibly to deter predation or prevent desiccation while maintaining gaseous exchange. The incorporation of minerals into or over capsule surfaces has Steenstrupia 30 (1): 115–125.

occurred in a number of gastropod families (e.g., eggs of ampullariids and terrestrial pulmonates; sand collars of naticids). However, little work has been done since to examine the morphology and surface structure of neritid egg capsules. The hatching mechanism is also not known. While substantive anatomical, behavioural and ecological work has been done on neritids (Berry et al. 1973; Chelazzi 1982; Estabrooks et al. 1999; Fretter 1946, 1965; Garrity & Levings 1981; Haynes 1991; Houston 1990; Hughes 1971; Komatsu 1986; Starmühlner 1976), their species-level taxonomy, particularly of those in Southeast Asia, is relatively poorly defined (Spencer et al. 2007). Egg capsule structure could provide critical information needed to distinguish closely related species. Tropical shores generally

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support a number of Nerita species in the same habitat, and ecological studies might benefit if egg capsules of various species can be differentiated visually, if not in the field, at least under a microscope. This preliminary study attempts to show that each Nerita species in Singapore has a unique egg capsule morphology. The functional morphology of the egg capsule is also explored in relation to hatching.

MATERIAL AND METHODS The egg capsules of seven species of Nerita, and one species each of Clithon and Dostia, from Singapore were examined: Nerita albicilla L., 1758; N. chamaeleon L., 1758; N. balteata Reeve, 1855 (=articulata Gould, 1847; = birmanica Troschel, 1877; = lineata Gmelin, 1791; see Cernohorsky 1978: 42); N. planospira Anton, 1839; N. polita L., 1758; N. squamulata Le Guillou, 1841 (= N. histrio Gmelin, 1791?); N. undata L., 1758; Clithon oualaniensis (Lesson, 1831) and Dostia violacea (Gmelin, 1790). Shells of most species are illustrated in Tan & Chou (2000) and Tan & Clements (2008). Animals were collected by hand from rocky shores, mangroves and estuaries in Singapore during low tide. Several individuals of each species were kept in small, plastic tanks submerged completely in flow-through or recirculating seawater aquaria to encourage egg capsule deposition. Daily checks were made for capsules, which were carefully removed from the tank walls using a razor blade and examined under stereo- and compound microscopes. A simple polarizing microscope was used to determine if spherulites showed birefringence. Egg capsules for scanning electron microscopy were either air-dried or fixed in 3% buffered glutaraldehyde adjusted to be osmotically compatible with seawater and kept in 70% ethanol. Specimens were coated either with gold or platinum. Capsules were also kept in seawater at room temperature to observe how larvae emerge from within. It was possible to count the number of embryos through the membranous base if the capsules were removed from the substratum carefully after they are laid. Where possible the

initial number of embryos present and the number of larvae that develop inside the egg capsules were tracked during development. Reinforcement bodies, calcareous spherulites and composite nodules all refer to the variously shaped calcium carbonate crystals or sand particles that characterize the surfaces of neritid egg capsules.

RESULTS Species descriptions

(see Table 1 for comparative summary)

Nerita albicilla

(Figs 1A–B, 2F, 3A–B; see also Knudsen 1992)

Egg capsules are translucent white and oval in outline, up to 2.1 mm in diameter, and 300 µm in height. The external surface of the cap is scattered evenly with lens-shaped spherulites each between 50–70 µm in diameter (Fig. 1A), which are characteristically birefringent under polarized light (Fig. 1B). The spherulites are arranged closely, sometimes in contact with each other, up to 20 µm apart in a single layer partially embedded in a light yellow, seemingly homogenous organic matrix (Fig. 3A, B). This is consistent across the entire cap surface, with little or no differentiation in the arrangement or size of the spherulites between those at the central region and towards the edge of the cap. The surface of the spherulites appears smooth, and the inside is solidly packed with undetermined elongate crystals oriented perpendicular to the surface (Fig. 2F). Nerita balteata (Figs 1C, 3D)

Egg capsules of this species are some of the largest amongst its congeners examined, reaching up to 4 mm in diameter, and 500 µm in height. They are often deposited on hollowed out mangrove tree bark and are thus flush with the substratum surface. The external surface of the cap is covered with spherulites which are approximately spherical in shape and comprising two distinct size groups (t-test, P < 0.001, n = 20). The smaller, smooth-surfaced but hollow spherulites are be-

neritid egg capsules

A

B

C

D

E

F

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Fig. 1. Surface features of Nerita egg capsules. A. Nerita albicilla. B. Nerita albicilla. Egg capsule spherulites in situ under polarized light (530 nm). C. Nerita balteata. D. Nerita chamaeleon. E. Nerita chamaeleon. Egg capsule spherulites in situ under polarized light (530 nm). F. Nerita planospira. Egg capsule surface is devoid of spherulites and comprised only of an organic matrix with fine processes extending beyond the capsule surface. – Scales: A = 100 µm; B–C, E–F = 50 µm; D = 10 µm.

tween 10 and 20 µm in diameter, whilst the larger, solid spherulites are between 30 and 70 µm in diameter with flattened hexagonal crystals on the surface (Fig. 1C, 3D). These spherulites do not appear to be birefringent under polarised light.

Nerita chamaeleon (Figs 1D–E)

Egg capsules dirty-white, relatively small, domeshaped, measuring up to 2.4 mm in diameter and 500 µm in height. The external surface of the

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A

B

C

D

E

F Fig. 2. A–E. Surface features of Nerita egg capsules. A. Nerita polita. B. Nerita squamulata. C. Nerita undata. D. Dostia violacea. E. Clithon oualaniensis. – F–G. Spherulite interior morphology. F. Nerita albicilla. G. Nerita polita. – Scales: 10 µm.

G

cap comprises very fine, oval, dumbbell-shaped or spherical grains of varying sizes ranging between 3 and 33 µm. Spherulites are closely packed together and the matrix is completely hidden. Crystals have a rounded outline and are birefringent under polarized light.

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neritid egg capsules

Nerita planospira

Nerita squamulata

Egg capsules translucent white, oval in outline reaching 2.0 mm in diameter and 375 µm in height. The external surface of the cap comprises fine branching processes continuous with underlying granular organic matrix, forming a fuzzy surface layer. Reinforcement bodies were absent from a total of ten capsules examined, and no crystal sac was found in five adult females dissected.

Egg capsules white and often round in outline, measuring up to 2.7 mm in diameter and 250 µm in thickness. These capsules are relatively larger but thinner than those of closely related congeners N. undata and N. chamaeleon. The cap comprises very fine, approximately spherical (larger > 20 µm) and rectangular (smaller < 10 µm) crystal grains of varying sizes ranging between 5 and 33 µm tightly packed together. The surfaces of these angular grains have a roughened texture etched with irregular patterns. The larger spherulites display birefringence under polarized light, while the smaller crystals show little or no birefringence.

(Figs 1F, 3C)

(Fig. 2B)

Nerita polita (Figs 2A, G)

Egg capsules white, small, somewhat hemispherical with a tall, rounded cap measuring up to 1.8 mm in diameter and 400 µm in height. The cap comprises uniform, rice grain-shaped spherulites closely packed together. The spherulites are not birefringent under polarized light. The spherulite surface comprises small, flattened hexagonal crystals somewhat reminiscent of nacre. According to Bandel (1991), the spherulites of this species are composed of calcite.

Nerita undata

(Figs 2C, 4A–D, 5A–D)

Egg capsules large, up to 3.2 mm in diameter, white, with a height of up to 650 µm. The cap comprises small, spherical spherulites 20–60 µm in diameter that display slight birefringence under polarized light. These spherulites are loosely

Table 1. Egg capsule characteristics of some Singapore Neritidae. Capsule diameters and heights refer to maximum dimensions as measured under a light microscope. Cap wall thickness was measured from cut matrix surfaces devoid of spherulites to the roof of the capsule chamber as observed through a scanning electron microscope. Wall thickness can be considerably thinner under individual spherulites. Species

Capsule diameter (mm)

Capsule height (µm)

Cap wall thickness (µm)

Spherulite shape and size range (µm)

Number of embryos

Nerita albicilla

1.7–2.1

300

26–28 (n=3)

lens-shaped, 50–75

60–121 (n=5)

Nerita balteata

3.4–4.2

500

40–42 (n=3)

spherical, bimodal in size, small: 8–28,large: 30–63

153, 154 (n=2)

Nerita chamaeleon

1.8–2.4

500

17–20 (n=3)

oval or spherical, 3–33

24–65 (n=16)

Nerita planospira

1.7–2.0

375

23–45 (n=3)

absent

100 (n=1)

Nerita polita

1.4–1.8

400

11–14 (n=3)

oval, 45–50

31–60 (n=50)

Nerita squamulata

2.1–2.7

250

14–17 (n=3)

spherical or rectangular, with mostly angular edges, 5–33

18–25 (n=5)

Nerita undata

2.8–3.2

650

41–47 (n=3)

spherical, 10–60

15–37 (n=50)

Dostia violacea

1.4–1.6

250

20–22 (n=3)

high density of foreign material incorporating sand grains and diatoms, irregular, 5–100

40–62 (n=5)

Clithon oualaniensis

1.0–1.1

250

10–13 (n=3)

medium density of foreign material incorporating sand grains and diatoms, irregular, 5–100

41, 51 (n=2)

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A

B

C

D

Fig. 3. Nerita egg capsule organic matrix. A. Nerita albicilla. Capsule surface with spherulites removed. B. Nerita albicilla. Cross-section of cap. C. Nerita planospira. Cross section of cap showing branching surface processes. D. Nerita balteata. Cross-section of cap. – Scales: 10 µm.

arranged in the central region of the cap such that the matrix is visible (Fig. 2C, 4A), whilst they are densely packed near the edges. There are no marked differences in their size distribution of the spherulites across the cap surface. Spherulite surfaces are highly irregular. The inside surface of the cap has a broad, flattened rim (Fig. 4B) that fits over a corresponding surface on the tambour (Fig. 4C). Microscopic foldings of the surface (Fig. 5B, C) can be discerned on the surface of the tambour rim, which are themselves crossed by narrow, flattened fibres. These fibres are also present on the cap rim surface (Fig. 4D). The inside edges of both cap and tambour bears a protuberance, at which the cap pivots when it separates from the tambour to release the larvae. A strut-like support (absent in the tambour) extends from the protuberance of the cap towards the centre (Fig. 4B).

Dostia violacea (Figs 2D, 5E)

Egg capsules dirty white, small, oval in outline with a distinct border about 100 µm wide around the rim of each intact capsule as seen from above. The external surface of the cap comprises sand grains and diatom frustules of varying sizes ranging between 5 and 100 µm embedded in a uniform matrix. The cap surface is generally indistinguishable from the capsules of Clithon oualaniensis (see Fig. 2E and below). In intact but open capsules (i.e., immediately after larvae are released), the basal portion of the capsule, i.e., the tambour, adhering to the substratum is also oval in outline with a white, uniformly narrow raised rim about 100 µm wide. The enclosed area inside is concave and lined with a thin, transparent membrane that adheres to the substratum (in

neritid egg capsules

A

B

C

D

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Fig. 4. Nerita undata. A. Surface view of cap. B. Inside of cap (roof of egg capsule chamber) showing strut-like support (arrow) extending from internal edge of rim to centre of cap. C. Detail of cap rim, showing fibril orientation. D. Fibrils on surface of cap rim. – Scales: A–B = 100 µm; C = 10 µm; D = 1 µm.

this case, a dead mangrove tree trunk). A thin, convex, transparent membrane enclosing clear liquid at slight positive pressure in turn encloses the entire basal region of the capsule. The dorsal cover of open capsules is attached to the lower tambour at a point that presumably acts as a hinge. The hinge is distinguished as a thinner, raised region on the basal rim. The dorsal cover also has a thin, convex, transparent membrane attached to its underside, which in turn encloses a transparent, liquid medium. This liquid could be used to exert positive osmotic pressure within the capsule when the larvae are ready to hatch. Larvae are probably enclosed between the two transparent convex-shaped membranes in intact capsules.

Egg capsules of Dostia violacea laid on bark of broken mangrove branches lying on the mangrove floor were allowed to hatch in the laboratory. Upon maturity, the cap of the capsule became partially detached and swung away from the lower tambour (Fig. 5E), releasing the larvae within. A single point of attachment between the cap and the tambour was located at a slightly thickened region of an otherwise uniform rim on the opposing edges. This opening action appeared to be effected by distension of the thin transparent membranes on the inner walls of cap and tambour exerting positive pressure on each other. Essentially, the ballooning of the two membranes under pressure pushes the larvae out from within the capsule.

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A

B

C D

E

Fig. 5. A–D. Nerita undata. A. View of egg capsule and embryos inside, as seen through the transparent capsule base or tambour. B. Inside of tambour, showing the nearly featureless surface. C. Detail of tambour rim, showing folds on fibrils. D. Underside of tambour in contact with the substratum; numerous vesiculations are visible, presumably containing adhesives that provide the necessary bond between tambour and the substratum. – E. Dostia violacea. Opened egg capsules (with one capsule still intact). Opposing inflating membranes in the cap and tambour provide the necessary mechanism for the cap to swing away from the base, releasing the veligers inside. – Scales: A–B, D = 100 µm; C = 10 µm; E = 1 mm.

Clithon oualaniensis

ranging between 5 and 100 µm embedded in a uniform matrix.

Egg capsules small, dome-shaped with an oval outline, reaching up to 1.1 mm in greatest diameter. The external surface of the cap comprises sand grains and diatom frustules of varying sizes

Other observations

(Fig. 2E; see also Knudsen 1992)

There was considerable intra- and interspecific variation in the number of embryos inside each

neritid egg capsules

capsule. Numbers ranged between the low tens (e.g., Nerita chamaeleon, N. undata) to more than a hundred (e.g., N. balteata, N. planospira) in each capsule (Table 1). These nearly all mature into planktotrophic larvae based on laboratory observations and/or embryo size. Reduction in numbers during development by way of nurse eggs was not observed. The embryos appear to be enclosed in a delicate membranous chamber, which breaks open when they are ready to be released from the capsule. The mechanism of release appears to be effected by positive internal pressure exerted by the distension of two thin transparent membranes on the inner walls of cap and tambour (see Fig. 5E). Based on laboratory observations of the hatching of Dostia violacea larvae, the ballooning of the two membranes under pressure causes the larvae to be pushed out from within the capsule.

DISCUSSION All capsules examined in this study comprise a shallow, thin walled tambour (Andrews 1935), covered by a thick-walled cap, enclosing the embryos within. The lower tambour is mostly a thin membrane that adheres to the substratum, but has a distinct thickened, raised rim. The upper cap, which also has a thickened rim, is a more robust structure with a substantially thicker wall (8–40 µm) throughout. Cap and tambour are firmly attached to each other along their rims. They separate partially or completely when the larvae are ready to hatch. The external surface of the cap is structurally complex, with reinforcement material comprising either calcium carbonate spherulites manufactured internally or material derived from consumed items such as sand grains, diatoms and foraminifera. Such material is embedded partially in a layered organic matrix. Spherulites up to 100 µm in size or irregular-shaped particles of external origin are arranged regularly or haphazardly over the cap surface. In some species the reinforcement bodies tend to be larger at the outer edges of the cap but smaller towards the centre. All but one Nerita species examined in this study laid egg capsules reinforced with

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spherulites. In contrast, the brackish water species Dostia violacea and Clithon oualaniensis deposited capsules containing sand grains and diatoms. Members of freshwater species in the genera Theodoxus, Neritina and Septaria all deposit capsules reinforced with foreign material, presumably due to reduced calcium levels in the environment, while Dostia and Clithon do not live in calcium-limiting environments. Whether species of different genera that share these disparate habitats are phylogenetically each other’s closest relatives, or whether this character evolved independently more than once, requires future investigation. The results of this study clearly show that the egg capsules of Nerita species in Singapore can be distinguished based on spherulite morphology. Andrews (1937) earlier pointed out that spherulites on the egg capsules of three Caribbean species of Nerita (N. peloronta, N. versicolor and N. tessellata) were different. Indeed, spherulite shape and size appear to be relatively consistent within members of each species. Those of N. albicilla from India (Natarajan 1957), Hong Kong, China and N. squamulata (as N. histrio) from Darwin, northern Australia observed by Knudsen (1992, 1997) are comparable with Singapore specimens, despite their geographical separation. Unfortunately the illustration provided by Knudsen (1992) of the spherulites on the egg capsule of N. chamaeleon is not sufficiently detailed to distinguish between this species and N. squamulata. The shell illustrated by Knudsen (1992) appear to be of the latter species. The mineralogy of the spherulites was not determined in this study, but the nature of calcium carbonate (whether calcitic or aragonitic in composition; Bandel (1991) found calcitic spherulites in Nerita polita) could be another character that may assist in distinguishing species. In the mangrove species Nerita planospira, spherulites are absent altogether, and the external surface of the egg capsule comprises fine outgrowths of the organic matrix. The crystal (or reinforcement) sac from which the spherulites are derived, was also lacking in adult females of this species. Its basal position on the molecular phylogenetic tree of Nerita species constructed from mitochondrial and nuclear markers (Frey & Vermeij 2008) offers a possible explanation for this observation.

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Amongst the neritopsine families, only members of the Neritidae are known to possess crystal sacs and lay reinforced egg capsules (Andrews 1935, Fretter 1946). In contrast, a crystal sac is absent in the Neritiliidae (Kano & Kase 2002) although egg capsules are reinforced with sand or diatoms derived externally (Kano & Kase 2002, Kano et al. 2001). The crystal sac is also absent in Phenacolepas (Fretter 1984). Little is known of the method of reproduction in the terrestrial Helicinidae (e.g., Richling 2004) and Hydrocenidae (Haase & Schilthuizen 2007), but members of these two poorly researched families appear to lack a crystal sac, although eggs are calcified. The anatomy and egg capsules of Neritopsis radula, the sole member of the family Neritopsidae, are also unknown. The origins of the calcium carbonate spherulites that accumulate in the crystal sac remain obscure. Andrews (1935, 1937) suggested that the spherulites were derived from the digestive gland, which made their way down the rectum and were somehow extracted by the crystal sac. Where and how the process of isolating spherulites from faecal matter occurs is unknown. Based on observations of this study, the extremely uniform contents of the crystal sac suggest an alternative, but as yet undetermined origin of the spherulites. Internally, the wall surfaces of both tambour and cap are optically smooth, and few surface characteristics could be discerned in all species examined. Cross-sections of fractured capsule caps seen under the scanning electron microscope reveal a uniform granular matrix just below the spherulite layer. Wall thickness varied between species, ranging from 10 µm (Clithon oualaniensis, Nerita polita) to 40 µm (N. balteata, N. undata) (see Table 1). The wall of the cap must balance the need for rigidity, to prevent the cap from collapsing over the embryos when the capsule is out of water, while providing porosity, to ensure sufficient but not excessive liquid and gaseous exchange. The granular matrix appears to become more compressed inwards to form a homogenous, non-granular matrix that also forms the characteristic rims of tambour and cap, where it is thickest. When the cap is separated from the base tambour, the rim surfaces of both tambour and cap bear distinctive ‘fibrils’ (Andrews 1935) that are oriented at an approximately

constant oblique angle to the rim edge. These ‘fibrils’ observed and illustrated by Andrews (1935) in Neritina reclivata are probably surface folds on the rim surface, as also seen on the rim surface of the tambour in Nerita undata (Fig. 5C). These folds are, in turn, crossed by very fine, overlapping ribbon-like fibres each about 1.5 µm wide (Fig. 5C) that are oriented perpendicular to the direction of the folds. Similar fibres are also seen on the inner half of the rim surface of the cap (Fig. 4D), although the folds are absent. The function(s) of these folds and fibres remain undetermined. The inner edge of the rims of both cap and tambour in all species examined is broken by a small protuberance jutting into the capsule interior. It probably serves as a reinforced pivot on which the weight of the cap rests when it separates from the base. The protuberance on the cap continues as a supporting curved edge that diminishes in size towards the centre of the cap. This may provide the necessary additional rigidity needed to prevent the cap from collapsing over the embryos when the capsules are out of water, given the additional weight of the reinforcement material present. Overall, the egg capsules of neritids exhibit surprising complexity in structure, despite their seemingly simple outward appearance. The capsules also show remarkable adaptation in protecting the embryos within from the rigours of the intertidal habitat.

ACKNOWLEDGEMENTS Dr Jorgen Knudsen’s seminal papers on gastropod egg capsules provided the initial stimulus for this study. We thank Mdm Loy Gek Luan, EM Unit, Department of Biological Sciences and Ms Tang Chui Ngoh, Department of Chemistry, respectively at the National University of Singapore for allowing us access to electron microscope facilities in their care. We also wish to extend are heartfelt appreciation to Dr Kathe Jensen, Zoologisk Museum, Copenhagen, who provided the rare opportunity to present this paper at the Symposium in celebration of the 90th birthday of Jorgen Knudsen. Constructive comments from two reviewers greatly improved the manuscript.

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