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Spawning and intra-capsular development of

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Article in Invertebrate Reproduction and Development November 2009

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Invertebrate Reproduction and Development, 53:3 (2009) 137143 137
Balaban, Philadelphia/Rehovot
0168-8170/09/$05.00 2009 Balaban

Spawning and intra-capsular development of Neritina zebra

(Bruguire, 1792) (Mollusca: Gastropoda: Neritidae)
under laboratory conditions

CRISTIANE XEREZ BARROSO1* and HELENA MATTHEWS-CASCON1,2

Laboratrio de Invertebrados Marinhos, Departamento de Biologia, Centro de Cincias, Universidade Federal do Cear,
Campus do Pici, Bloco 909, CEP 60455-760, Fortaleza, CE, Brazil
email: cristianexb@gmail.com
Instituto de Cincias do Mar, Universidade Federal do Cear, Av. Abolio, 3207,
Meireles, Fortaleza, CEP 60.165-08, CE, Brazil
email: hmc@ufc.br

Received 26 February 2009; Accepted 16 October 2009

Abstract
The gastropod Neritina zebra is distributed in Suriname and along the coast of Brazil, where it lives
on muddy bottoms of brackish water environments. The objectives of this study were to describe
the spawning of N. zebra and to investigate its embryonic and larval development under laboratory
conditions. The egg capsules are oval. The surface of the capsule in contact with the substratum is
flat, while the opposite surface is convex and covered by sand grains. One end has a small suture.
A thin membrane surrounds the eggs. Inside the capsules, the developing embryos and larvae are
surrounded by fluid (albuminous liquid). N. zebra has mixed development. It was not possible to
observe settlement and metamorphosis of veligers in the laboratory due to their death a few days
after hatching. In the 2007 experiment, the capsules opened, releasing veligers, after 21 days. In
2008, the capsules opened after 26 or 27 days after the salinity was reduced from 15 to 10 ppt.
N. zebra may be amphidromous, as are others of the same genus which inhabit environments with
a strong freshwater influence and have planktotrophic larvae.

Key words: Spawning, mixed development, amphidromous species

Introduction
Based on the larval ecology of benthic invertebrates,
three main types of development are recognized
(Thorson, 1950; Jablonski & Lutz, 1980): (1) plankto-
trophic: when the development involves a planktonic
larvae feeding on external food particles; (2) lecitho-
trophic: when a non-feeding larvae will metamorphose
after a period of a few hours or days in the plankton;
(3) intracapsular metamorphosis which occurs before
hatching. Another important distinction can be made
between species with an entirely planktonic devel-
opment and species that spend all or part of their
development in egg masses or capsules (planktonic,
encapsulated nonplanktonic and mixed development,
respectively) (Pechenik, 1979).
Mixed development incorporates aspects of encap-

*
Corresponding author.
138 C. Xerez Barroso and H. Matthews-Cascon / IRD 53 (2009) 137143
sulated and planktonic developmental strategies with
only a portion of the pre-juvenile period being
encapsulated. The energy expenditure associated with
encapsulation appears to be high, which suggests that it
must have substantial survival value in mixed life
histories (Pechenik, 1979; Poulin et al., 2001; Chaparro
et al., 2005).
Neritids have internal fertilization and encapsulate
their eggs after fertilization, allowing the protection of
offspring until at least the veliger stage (Aktipis et al.,
2008).
Neritina zebra (Bruguire, 1792) (Gastropoda: Neri-
timorpha: Neritidae) occurs in Suriname and along the
coast of Brazil from Par to Cabo Frio (Rio de Janeiro)
(Rios, 1994). It is a common species on muddy bottoms
of brackish water environments (Rios, 1994). Although
there are a few studies on shell characteristics, taxo-
nomy and geographic distribution of N. zebra (Baker,
1924; Matthews-Cascon et al., 1990; Mienis, 1991;
Rios, 1994), little is known of its biology. This study
describes for the first time, the spawning of N. zebra and
its embryonic and larval development under laboratory
conditions.
Materials and Methods
Area of study, observation of spawning in the field and
collection of specimens
This study was conducted in a mangrove area in the
Cear River Estuary, Cear, Northeast Brazil (S
03E44N11.1O W 038E37N23.6O) (Fig. 1). The Cear
River Estuary covers an area of approximately 5 km of
mangrove. It has an equatorial climate with maximum
rainfall during the months of March, April, May and
June. In the estuary, the salinity varies seasonally and is
governed primarily by precipitation.
According to FUNCEME (Fundao Cearense de
Meteorologia e Recursos Hdricos), the total precipi-
tation of the study area from March 2007 to March 2008
varied considerably, reaching a maximum of 320.4 mm
in March 2008 and a minimum in August and November
2007, when there were no records of rain. The salinity
was also measured during this period in the sample site,
except in November 2007. The salinity reached a
maximum of 34 in October 2007 and a minimum of 5 in
March and June 2007.
Observations of reproductive activity (absence or
presence of egg capsules) of Neritina zebra were made
monthly during spring tides from March 2007 to March
2008. In March 2007 and March 2008, 40 specimens of
N. zebra were collected manually. The shells ranged in
length between 13.85 and 20.64 mm. The specimens
were placed in plastic containers and taken to the
laboratory for further observations of egg capsule
deposition and embryonic and larval development.
Water for the experiments was collected at the sampling
site.
Laboratory procedures
In the laboratory, the animals were placed in 20
plastic boxes (400 ml), two to each box to maximize the
number of capsules per day. These boxes were placed in
an aquarium of 60 L, with water from the place of study,
held under constant aeration and at temperatures
between 26C and 28C. Salinity was maintained at the
level of the field site on the day when the animals were
collected. In the experiment conducted in March 2007
the salinity was 5, while in March 2008 it was 15.
Description of spawning
The general structure of the egg capsules was
recorded and each one was measured with the aid of a
graduated slide with accuracy of 0.1 mm and a light
microscope. The length of the capsule was considered as
the greatest distance between the ends of the capsule and
the width was considered the shortest distance. The
number of eggs in each capsule was also recorded.
Monitoring of embryonic and larval development
For the daily monitoring of the development of
N. zebra, one box was chosen in March 2007 and 12 in
March 2008. The boxes chosen were those that con-
tained more than 30 capsules laid on the same day. The
selected boxes were then placed in 5 L aquaria and the
adults removed to avoid predation of the capsules by the
parents.
Each day, a capsule was removed for observation
with a microscope (Olympus CH2) with a digital camera
(Sony Cyber-shot 5.1 Megapixels) to register the
embryonic and larval stages. Because the capsules are
not transparent and have large numbers of eggs, it was
necessary to open the capsules to observe development.
The material used in the present study was deposited
in Coleo Malacolgica Prof. Henry Ramos Matthews
(CMPHRM) of Universidade Federal do Cear (UFC)
(CMPHRM 2571 and CMPHRM 2572).
Results
Observation of spawning in the field
In the field, egg capsules were deposited on any
available substratum such as wood, leaves, shells of
molluscs, including shells of living individuals of
Neritina zebra. A large number of egg capsules were
 C. Xerez Barroso and H. Matthews-Cascon / IRD 53 (2009) 137143 139

Fig. 1. Area of study: Cear River Estuary, metropolitan region of Fortaleza

(Cear), northeast coast of Brazil.

found in the pneumatophores of Avicennia sp. Spawns
were observed in the months of March, April, July,
August and September 2007 and January, February and
March 2008.
Reproductive behavior and description of egg capsules
During mating the male stays over the right side of
the female, inserting his penis below her mantle edge
and transferring the spermatophore. The egg capsules
are then transferred by the females foot to the walls of
the boxes or onto the shell of another adult.
The egg capsules have a yellowish color when
deposited, becoming darker as development progresses.
They are oval 1 to 1.5 mm long and about 1 mm wide.
The surface of the capsule in contact with the sub-
stratum is flat, while the opposite surface is convex. The
convex surface is covered by sand grains of different
sizes, from the sac of reinforcement (Figs. 2A and 2B).
Over time, the capsules laid in the laboratory did not
have sand, due to the absence of sand in the aquaria. The
capsules also have a thin membrane surrounding the
eggs (Fig. 2C) and a small suture, at one ends (Fig. 2D).
Inside the capsules, the embryos and larvae are
surrounded by a fluid (albuminous liquid or albumen).
Each capsule had, on average, 68 eggs (ranging from
32106, n = 60, SD = 14.62).
Embryonic and larval development
The egg capsules laid in the laboratory permitted the
study of intracapsular development of N. zebra from
cleavage to the release of veliger larvae in the water, as
N. zebra has mixed development. It was not possible to
observe settlement and metamorphosis of veliger in the
laboratory, due to death of the free-swimming veligers a
few days after hatching. There were no indications of
the presence of nurse eggs or death of embryos inside
the capsule.
At salinity of 5 (experiment developed in 2007), the
capsules opened, releasing veligers, after 21 days. At
salinity of 15 (experiment developed in 2008), develop-
ment was slower than at 5, and after 25 days, despite
having well formed veligers, these did not hatch. To
140 C. Xerez Barroso and H. Matthews-Cascon / IRD 53 (2009) 137143

Fig. 2. Egg capsules of Neritina zebra. A) General appearance of the capsules showing its two surfaces, the flat surface (b),
which adheres to the substrate, and the convex surface (ps), which is impregnated with sand grains. Scale bar: 77 m.
B) Detail showing the grains of sand embedded in the upper surface of the capsule. Scale bar: 48 m. C) Detail showing the
membrane (m) surrounding the eggs inside the capsule. Scale bar: 70 m. D) Detail showing the suture (s) in the egg capsule,
next to one of its ends. Scale bar: 60 m.

induce hatching of these larvae, we decreased salinity
from 15 to 10. The capsules opened 24 or 48 h after this
salinity change releasing the veligers.
Embryonic and larval stages observed in the labora-
tory during the years 2007 and 2008 are given in
Table 1. There were differences in the duration of
embryonic and larval stages between the two salinities
(Table 1, Fig. 3AH). There was synchronized devel-
opment within and among the capsules of the twelve
boxes of year 2008, except for Day 1, which had
embryos with one, two or four cells within the same
capsule.
Discussion
Among the species of Neritidae, the capsules differ
in shape, size and, especially, the nature and size of
particles used in the reinforcement of capsules. The
particles used vary depending on the characteristics of
the substrate on which the animals feed (Andrews,
1933). While in this study, the capsules of N. zebra
covered only with sand grains of different sizes,
different materials to reinforce were found in other
species, such as: sponge spicules, limestone fragments
and diatom frustules. The differences in shape and size
of the capsules are due to the different ways in which
they are manipulated after leaving the oviduct
(Andrews, 1933; Fretter & Graham, 1962).
In this study, it was observed that N. zebra has
planktonic larvae. Although all species of the family
Neritidae deposit their eggs in characteristic capsules,
there are differences in reproductive strategies between
species. Three types of reproductive behavior have been
described: (1) freshwater species such as Theodoxus
fluviatilis and Theodoxus danubialis (Pfeiffer, 1828),
 C. Xerez Barroso and H. Matthews-Cascon / IRD 53 (2009) 137143 141

Table 1. Development of Neritina zebra. Duration (days) of the embryonic and larval stages of N. zebra in experiments
conducted in the laboratory in the years 2007 and 2008

Event 2007 2008

(day) (day)
Fertilized eggs (133.2 m), showing the first cleavages; uncleaved eggs and embryos containing 1 1
two or four cells were found in a single capsule (Fig. 3A)
Spiral holoblastic cleavage; blastula showing typical molluscan cross (Fig. 3B) 2 2
Embryos with several micromeres at the animal pole and macromeres at the vegetal pole (Fig. 3C) 3 3
Gastrula stage (cells more elongated) (Fig. 3D) 4 45
Pre-veliger larvae (pretorsional), vibratory movement (without displacement) for the first time; 56 68
presence of pre-velum and thin shell (Fig. 3E)
Veliger larvae (postorsional); small bilobed velum; presence of yolk, eyes as dark pigmented points; 78 910
lateral vibratory movement, with limited movement capacity (Fig. 3F)
Veliger larvae; thin and transparent operculum observed for the first time; statocysts (yellow 920 1125
pigmented points) observed in the larval foot with circular and fast movements, extended movement
capacity (Fig. 3G)
Open capsules, release of veligers in aquarium; 21 2627
Observation of free-swimming veligers, moving in circles (Fig. 3H) 2223 29

Fig. 3. Embryonic and larval development of Neritina zebra in experiments conducted in laboratory in the years 2007 and
2008. A) Fertilized eggs, showing the first cleavages. Scale bar: 30 m. B) Spiral holoblastic cleavage; blastula with typical
molluscancross. Scale bar: 15 m. C) Embryos with several micromeres (mi) at the animal pole and macromeres (ma) at the
vegetal pole. Scale bar: = 10 m. D) Gastrula stage. Scale bar: = 15 m. E) Pre-veliger larvae (pretorsional). Scale bar: 25 m.
F) Veliger larvae (postorsional). Scale bar: 35 m. G) Veliger larvae. Scale bar: 15 m. H) Free-swimming veliger larvae.
c, larval shell; e, estatocyst; ma, macromeres; mi, micromeres; mv, visceral mass; o, eye; op, operculum; p, foot; v, velum.
Scale bar: 65 m.
142 C. Xerez Barroso and H. Matthews-Cascon / IRD 53 (2009) 137143
and Neritina virginea (marine species), which hatch as
miniature adults, a crawling snail (intracapsular meta-
morphosis), (2) estuarine species such as Clithon
spinosus Sowerby, 1825, Clithon retropictus (von
Martens, 1879), Neritina granosa Sowerby, 1825,
Neritina listeri (Pfeiffer, 1840), Neritina afra Sowerby,
1843, Neritina latissima Broderip, 1833 and Neritina
canalis Sowerby, 1825, which hatch as planktotrophic
veligers larvae, and they can show amphidromic
behavior and (3) marine species such as Smaragdia
viridis (Linnaeus, 1758), Nerita atramentosa Reeve,
1855, Nerita plicata Linnaeus, 1758, Nerita albicilla
Linnaeus, 1758, Nerita scabricosta Lamarck, 1822 and
Nerita funiculata Menke, 1851 which hatch as long
lived planktotrophic veligers larvae (Andrews, 1935;
Bandel & Kowalke, 1999; Bunje, 2007; Crandall et al.,
2008; Ford, 1979; Hurtado et al., 2007; Matthews-
Cascon & Martins, 1999; Myers et al., 2000; Orton &
Sibly, 1990; Pyron & Covich, 2003; Resh et al., 1992;
Scheltema, 1971; Schneider & Lyons, 1993; Shigemiya
& Kato, 2001; Waters et al., 2005).
In this study, it was not possible to determine
whether N. zebra has amphidromous behavior like
estuarine neritids of the same genus which have
planktotrophic larvae and are representative examples of
amphidromous animals. Amphidromous animals spawn
in the environment where they live and their veligers
emerge from the capsules going to the ocean, where they
develop as planktotrophic veligers until metamorphosis,
when they are brought back to the estuarine environ-
ment (Bandel & Kowalke, 1999; Kano & Kase, 2003;
Strong et al., 2008).
N. zebra, like N. granosa, N. latissima and N.
canalis, also inhabits environments with strong fresh-
water influence. It is possible, therefore, that N. zebra is
an amphidromous species, as others of the same genus.
The difficulties associated with simulating an estuarine
environment in the laboratory, with its cycles of tides
and the migration of larvae from the river to the ocean
and the ocean to the river, could account for the inability
of N. zebra to complete embryonic and larval
development. However, authors who claim that some
species of Neritidae exhibit amphidromous behavior
(Andrews, 1935; Bandel & Kowalke, 1999; Ford, 1979;
Kano & Kase, 2003; Myers et.al, 2000; Shigemiya &
Kato, 2001; Pyron & Covich, 2003; Resh et al., 1992;
Schneider & Lyons, 1993; Strong et al., 2008), do not
explain how long the larvae live in the plankton, how
long the larvae can survive in the ocean, how the larvae
return to the mouth of the rivers and what their settle-
ment. This lack of information makes the understanding
of this reproductive strategy in these molluscs difficult.
The development and the hatching of intracapsular
veligers of N. zebra seem to be affected by salinity,
since the intracapsular development was fastest in the
egg capsules subjected to lower salinity (first experi-
ment) and the capsules in the second experiment only
opened after the salinity was reduced. In an experiment
describing the reproduction of Nerita atramentosa, the
larvae only emerged from the capsules when these were
artificially opened (Lesoway & Page, 2008). This
demonstrates that the opening of the capsules is linked
to some external stimulus. The hatching of juveniles
may be postponed until environmental conditions
become favorable.
Studies on the biology of species are important for
the systematic groups and to understand the ecological
role of various species in different ecosystems. This
study has added important information on the develop-
ment of intracapsular N. zebra, a species hitherto little
studied.
The egg capsules and development of intracapsular
N. zebra were characterized for the first time. Through
this research, we concluded that this species has a mixed
development, with development, probably more influ-
enced by the salinity of the environment where they live.
This study therefore provides important infor-mation for
a better understanding of the biology of N. zebra and
subsequent classification within the family Neritidae.
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