Early development of Betta splendens under stereomicroscopy and scanning electron microscopy

Abstract

Betta splendens
is a very important ornamental species. The current paper describes the embryonic and larval development of
B. splendens
under stereomicroscopy and scanning electron microscopy. Eggs and larvae from natural spawning were collected at different developmental stages at previously established intervals and analysed. The eggs of
B. splendens
are yellowish, clear, spherical, demersal, translucent and telolecithal with a large amount of yolk. Between 0–2 h post-initial collection (hpIC), the eggs were at the egg cell, first cleavage and morula stages. The blastula stage was identified at 2–3 hpIC and the early gastrula phase was observed at 3–4 hpIC with 20% epiboly, which was finalized after 13–18 hpIC. When the pre-larvae were ready to hatch, the appearance of somites and the free tail were observed, at 23–25 hpIC. At 29 hpIC, the majority of larvae had already hatched at an average temperature of 28.4 ± 0.2°C. The newly hatched larvae measured 2.47 ± 0.044 mm total length. The mouth opened at 23 h post-hatching (hPH) and the yolk sac was totally absorbed at 73 hPH. After 156 hPH, the heart was pumping blood throughout the entire larval body. The caudal fin, operculum and eyes were well developed at 264 hPH. When metamorphosis was complete at 768 hPH, the larvae became juveniles. The current study presents the first results about early development of
B. splendens
and provides relevant information for its reproduction, rearing and biology.

Full text

Zygote: page 1 of 10 c
Cambridge University Press 2013
doi:10.1017/S0967199413000488
Early development of Betta splendens under stereomicroscopy and
scanning electron microscopy
Fernanda Nogueira Valentin2,Nivaldo Ferreira do Nascimento2,Regiane Cristina da Silva2,
João Batista Kochenborger Fernandes2,Luiz Gustavo Giannecchini and Laura Satiko Okada Nakaghi1
Laboratório de Histologia e Embriologia do Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciências
Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, São Paulo, Brazil; and Centro de Aquicultura da
Universidade Estadual Paulista (CAUNESP), Jaboticabal, São Paulo, Brazil
Date submitted: 8.5.2013. Date revised: 20.6.2013. Date accepted: 9.9.2013
Summary
Betta splendens is a very important ornamental species. The current paper describes the embryonic and
larval development of B. splendens under stereomicroscopy and scanning electron microscopy. Eggs
and larvae from natural spawning were collected at different developmental stages at previously
established intervals and analysed. The eggs of B. splendens are yellowish, clear, spherical, demersal,
translucent and telolecithal with a large amount of yolk. Between 0–2 h post-initial collection (hpIC),
the eggs were at the egg cell, first cleavage and morula stages. The blastula stage was identified at 2–
3 hpIC and the early gastrula phase was observed at 3–4 hpIC with 20% epiboly, which was finalized
after 13–18 hpIC. When the pre-larvae were ready to hatch, the appearance of somites and the free tail
were observed, at 23–25 hpIC. At 29 hpIC, the majority of larvae had already hatched at an average
temperature of 28.4 ±0.2°C. The newly hatched larvae measured 2.47 ±0.044 mm total length. The
mouth opened at 23 h post-hatching (hPH) and the yolk sac was totally absorbed at 73 hPH. After
156 hPH, the heart was pumping blood throughout the entire larval body. The caudal fin, operculum
and eyes were well developed at 264 hPH. When metamorphosis was complete at 768 hPH, the larvae
became juveniles. The current study presents the first results about early development of B. splendens
and provides relevant information for its reproduction, rearing and biology.
Keywords: Eggs, Embryos, Fish, Larvae, Ontogeny, Reproduction
Introduction
Betta splendens, widely cultivated as an ornamental
fish, is a very important species for fish farming.
The betta fish has an auxiliary breathing organ
known as the labyrinth, which allows them to
breathe atmospheric oxygen and tolerate low levels
of dissolved oxygen in the water (Damazio, 1992;
Faria et al., 2006). Certain features of B. splendens,
such as colour, fin length and temperament, have
1All correspondence to: Laura S.O. Nakaghi. 2Laboratório
de Histologia e Embriologia, Departamento de Morfologia
e Fisiologia Animal, Faculdade de Ciências Agrárias e
Veterinárias, Universidade Estadual Paulista, Jaboticabal,
Via de Acesso Prof. Paulo Donato Castellane s/n, ZIP code-
14884–900, Jaboticabal–São Paulo, Brazil. Tel:/Fax: +55 16
3209 2654 (ext. 232). e-mail: laurankg@fcav.unesp.br
2Centro de Aquicultura da Universidade Estadual Paulista
(CAUNESP), Jaboticabal, São Paulo, Brazil.
been selected by breeders for centuries for ornamental
purposes and fighting. Therefore, in the wild, bettas
are less aggressive with less colouring and shorter fins
(Monvises et al., 2009).
Among the several Betta species, the best known
is Betta splendens (Faria et al., 2006; Monvises et al.,
2009). However, despite its commercial importance,
little information is known about its ontogeny and
it is during the early developmental stages, such as
the onset of exogenous feeding, that high mortality
rates are observed (Yúfera & Darias, 2007). Therefore,
study of the early developmental stages is necessary
to establish good production methods (Maciel et al.,
2010).
The embryonic developmental stage lasts from
fertilization of the oocyte by the sperm until hatching
of the larvae (Matkovic et al., 1985; Solnica-Krezel,
2005; da Rocha Perini et al., 2010). The larval
development stage begins with hatching and lasts
until metamorphosis is complete, when the larvae

2Valentin et al.
acquire morphological characteristics similar to those
of the adults and are known as juveniles (Kendall
et al., 1984). Meanwhile, a series of morphological
changes essential to survival are observed, such as
the development of fins, breathing and feeding (Osse,
1989; da Rocha Perini et al., 2010).
Stereomicroscopy and scanning electron microscopy
show structural differences during the development
of eggs and larvae (Paes et al., 2011), which provide
important information about betta biology. Therefore,
due to the importance and the lack of knowledge
about the ontogeny of this species, the current study
analyses the embryonic and larval development of
B. splendens using stereomicroscopy and scanning
electron microscopy.
Materials and methods
Site, animals and sampling
The experiment was carried out at the Ornamental
Fish Laboratory of the Aquaculture Center of UNESP
(CAUNESP), in Jaboticabal, São Paulo, Brazil. The av-
erage physico-chemical water parameters monitored
in the tanks were as follows: average temperature
28 ±0.2°C; dissolved oxygen 5.3 mg/L and conduct-
ivity 44 ␮S/cm2. Four animals (two males and two
females) were kept in individual 2-litre tanks equipped
with a water recirculation system for 10 days. After
this period, the females with visible oviducts were
transferred to the tanks with the males, where they
remained inside plastic cups with holes and were
released after a day to initiate reproductive behaviour.
Gamete release started after 3 days. This process was
slow and lasted approximately 3 h (the two couples
spawned and the eggs were pooled). The sampling
started at pre-established times: initial collection (IC;
as soon as eggs were observed in the nests), hourly
until 6 h post-initial collection (hpIC), every 3 h until
hatching, 1 h post-hatching (hPH), every 2 h until 19
hPH, every 4 h until 43 hPH, every 6 h until 91 hPH
and every 7 days until 936 hPH. Sampling consisted of
collecting 10 eggs and larvae at a time. From 72 hPH,
the larvae were fed with Artemia sp. twice daily. The
samples were fixed in 4% formaldehyde and 0.1 M
phosphate buffer, pH 7.4 and modified Karnovsky
(2.5% glutaraldehyde and 1.0% paraformaldehyde,
with 1.0 M cacodylate buffer and pH 7.2).
Microscopy
Eggs and larvae were examined under a LEICA
MZ8 stereomicroscope equipped with a LEICA DFC
280 camera, using the IM 50-LEICA software. Egg
diameter and total length of the larvae (n=10) were
measured in each sample. For scanning electronic
microscopy analysis, the samples were post-fixed in
1% osmium tetroxide for 2 h and washed in sodium
phosphate buffer. Subsequently, they were dehydrated
in graded series of ethanol at 30, 50, 70, 80, 90 and
95% concentrations plus three washes at 100% (10 min
each). Soon after, the samples were dried to the critical
point in a liquid CO2drier, mounted on a copper
grid, coated with gold–palladium ions, observed and
photographed under a scanning electron microscope
(JEOL-JSM 5410).
Results
Embryonic development
Betta splendens exhibited external fertilization and
partitioned spawning, with gamete release that
extended up to 3 h. Once spawning had finished, the
females were removed due to the aggressive behaviour
of the males.
Eggs from a single collection exhibit different
development stages due to partitioned spawning and
the pooling of eggs from two different spawning.
Therefore, eggs from the initial collection were
light yellow, spherical, translucent, telolecithal and
demersal. At this time, the eggs were at the egg cell,
first cleavage and morula stages, with an average
diameter of 1.08 ±0.038 mm (Fig. 1A–C). The early and
late blastula stages were identified between 2–3 hpIC
while the cells continued to divide mitotically (Fig. 1D,
E). The early gastrula was observed between 3–4
hpIC with 20% epiboly (Fig. 1F). Gastrula presented
between 30 and 50% epiboly at 5–6 hpIC (Fig. 1G).
In B. splendens, the blastoderm does not completely
surround the yolk; therefore, no yolk plug is formed
and approximately 50% of the yolk is covered. At 11–17
hpIC, thickening of the dorsal epiblast was observed,
which would give rise to the head of the embryo
(Fig. 1H).
At 21 hpIC, the head and tail were differentiated
and the first melanophores were present in the yolk,
found mainly near the ventral region of the embryo
(Fig. 1I). Over time, between 23–25 hpIC, the embryos
became pre-larvae. During this period, pairs of somites
occupied the entire notochord from the occipital to
the caudal region. The pre-larva was ready to hatch
and presented a free tail with strong and continuous
movements. This period, propagation and increase of
melanophores in the yolk toward the dorsal region of
the pre-larva was also observed (Fig. 1J). The hatching
began at 28 hpIC and the larvae presented poorly
pigmented eyes (Fig. 1K). After 32 hpIC, 90% of the
larvae had already hatched and after 38 hpIC, 100%
of the larvae had hatched. The total average length of
newly hatched larvae was 2.47 ±0.04 mm. Table 1 and

Early development of B. splendens 3
Figure 1 Main stages of the embryonic development of Betta splendens.(A) Egg cell; (B) 0–2 h post-initial collection (hpIC):
first cleavage; (C) 0–2 hpCI: morula; (D) 2–3 hpIC: blastula begins; (E) 2–3 hpIC: blastula ends; (F) 4–5 hpIC: beginning of the
gastrula with 20% of epiboly; (G) 6–9 hpIC: gastrula with 50% of epiboly; (H) 12–18 hpIC: gastrula at the end of epiboly; (I)21
hpIC: beginning of the formation of embryo and yolk pigmentation (arrow); (J) 24–26 hpIC: pre-larva, ready to hatch, showing
the cephalic and tail regions (arrows) and the presence of somites (arrowheads); (K) 29 hpIC: hatching of the larva and eye
pigmentation.
Fig. 1 show the main embryonic development stages of
B. splendens.
Larval development
Hatching
At the beginning of hatching, B. splendens measured
2.47 ±0.044 mm total length (TL) and presented
melanophores in the yolk at the antero-ventral axis.
At this moment, the newly hatched larvae showed a
closed mouth, large yolk sac, non-differentiated and
slightly pigmented eyes (Fig. 2A), a pectoral fin bud
and a well developed caudal fin (Fig. 3B).
At this stage, the larvae had low swimming capacity
and remained attached to the bubble nest under the
parental care of the male. For this reason, they had

4Valentin et al.
Table 1 Embryonic development of the Betta splendens at 28.4 ±0.2 °C
Time post-fertilization
Developmental stage (h) Note
Egg cell 0 Fig. 1A
First cleavage 0–2 Fig. 1B–Blastodisc divided to form two equal cells
Morula 0–2 Fig. 1C
Blastula begins 2–3 Fig. 1D
Blastula ends 2–3 Fig. 1E
Gastrula with 20% of epiboly 4–5 Fig. 1F–Blastoderm cells begin to spread over the yolk.
30–50% of epiboly 6–9 Fig. 1G–Germ ring epiboled ½of yolk sac
End of epiboly 12–18 Fig. 1F
Organogenesis begins 21 Fig. 1I–Beginning of embryo formation
Preparation for hatching 24–26 Fig. 1J, Fig. 3A–Pre-larva with free tail and the presence of somites
70% Hatching 29 Fig. 1K
adhesive glands that were identified in the dorsal
region of the head, just above the eye (Fig. 4A,B).
1–11 hPH
The olfactory cavity was formed after 1 hPH,
surrounded by mucus-producing cells (Fig. 4C), and
after 7 hPH it was deeper (Fig. 4D). It was observed
that the eyes were more pigmented; the notochord
more visible and the yolk volume reduced after 11
hPH (Fig. 2B). The melanophores were more evident
in the entire yolk and concentrated mainly on the
larval antero-ventral axis (Fig. 2B). At this stage,
the disappearance of the adhesive gland was also
observed.
11–43 hPH
Cilia were identified on the upper lip and the mouth
opening at 17 hPH (Fig. 5A), when the larva TL was
2.66 ±0.068 mm. At 43 hPH, the opercle covered the
gills (Fig. 3E). Despite the mouth opening was present,
we did not observe larvae feeding at this stage.
43–65 hPH
At 49 hPH the eyes and body were more pigmented
(Fig. 2D). At 65 hPH, a large amount of neuromasts
was observed in the lateral line and around the
eye (Fig. 5D–F). At this stage, a greater swimming
ability was observed, with the larvae displaying
morphological and sensory structures that enabled
greater mobility and perception of the surroundings.
65–264 hPH
At this stage, although the larvae still had yolk,
exogenous feeding had begun, as Artemia sp. nauplii
could be seen inside their bodies at 72 hPH (Fig. 6A).
The yolk was completely absorbed at 73 hPH, when
the larvae measured 3.20 ±0.176 mm in length. From
this phase onwards, the larvae exclusively obtained
food via exogenous feeding, by actively chasing
the nauplii (Artemia sp.) (Fig. 6B). After 86 hPH,
neuromasts were observed in the lower jaw (Fig. 5G).
At 156 hPH, the heart was pumping blood throughout
the entire larval extension (Fig. 6D). At 264 hPH, the
caudal fin was fully formed, with opercle and eyes well
developed (Fig. 3H).
264–768 hPH
At 432 hPH, the dorsal and anal fins were being
formed and the caudal fin rays were clearly evident
(Fig. 2F). Characteristics similar to the adults were
observed when larval TL was 17.24 ±2.064 mm, which
characterized the end of the larval stage (Fig. 2G),
after 768 hPH. From this point on, the animals are
considered juveniles. Table 2 describes the main stages
of larval development.
Discussion
Betta splendens are sedentary fish with parental
care, whose eggs adhere to the bubble nest. Betta
eggs are non-adhesive as, morphologically, they do
not present any of the structures that define this
characteristic such as zona radiata with hexagonal
pore canals, filaments, villous blood cells or gelatin
covers (Godinho & Godinho, 2003). However, the eggs
remain and develop in bubble nests constructed by
the male, who produces in his mouth a thick mucus
that consists of glycoproteins that help to maintain
the permanence of the bubbles in the nest (Kang &
Lee, 2010); thus, it is believed that this mucus may
also be linked to egg permanence in the bubble nest.
However, adhesive eggs are described for sedentary
fish such as Acestrorhynchus britskii,Acestrorhynchus
lacustris,Serrasalmus spilopleura (Rizzo et al., 2002),
Astronotus ocellatus (Paes et al., 2011) and Franciscodoras
marmoratus (Alberto Weber et al., 2012).

Early development of B. splendens 5
Figure 2 Larval development of Betta splendens.(A) newly hatched larva (29 h post-fertilization); (B) 11 h post-hatching (hPH);
(C)23hPH;(D)49hPH;(E)73hPH;(F) 432 hPH; (G) 768 hPH.
Betta splendens eggs do not have oil droplets and
are telolecithal. The yolk is most concentrated at the
vegetal pole while the organelles and cytoplasm are
concentrated in the animal pole (Kunz, 2004; Ninhaus-
Silveira et al., 2006). The eggs are demersal because
their specific gravity is greater than water (Godinho
& Godinho, 2003) and the animal pole is oriented
upward. This event occurs because the yolk sac has
a greater relative gravity than the blastodisc (Kunz,
2004).
Egg average diameter in the current study was
approximately 1.08 ±0.038 mm. This value is close to
the 0.8 mm reported by Watson & Chapman (2002) as
the average for ornamental species. The egg diameter

6Valentin et al.
Figure 3 Electron micrographs of Betta splendens.(A) 25 h post-fertilization (hPF): Pre-larva ready to hatch; (B) 29 hPF: newly
hatched larva; (C) 15 h post-hatching (hPH); (D)17hPH;(E)43hPH;(F)61hPH;(G)85hPH;(H) 264 hPH. FF: finfold. Y: yolk.
Arrowheads: mouth. Black arrows: pectoral fin. White arrows: opercle.
and quality are related to factors such as parental care,
breeder nutrition, ecological strategy, water quality,
photoperiod, animal welfare and genetic influence
(Brooks et al., 1997; Kolm & Ahnesjo, 2005). Parental
care is directly related to egg size: the larger the egg,
the greater the parental care (Kolm & Ahnesjo, 2005).
Usually, migratory fish exhibit another strategy, which
does not present parental care and produce a large
number of small eggs (Godinho et al., 2010).
The size and shape of the eggs may be important
for systematic and phylogenetic studies, as observed
in the identification of species of the genus Gobius
(Borges et al., 2003). Furthermore, it is also relevant
to identify spawning areas and to implement pro-
grammes to protect and preserve the species (Nakatani
et al., 2001).
The cleavage process, which consists of the division
of the egg into smaller cells named blastomeres, starts

Early development of B. splendens 7
Figure 4 Electron micrographs of Betta splendens showing some structures during development. (A,B) Newly hatch larvae,
adhesive glands. (C) 1 h post-hatching (hPH): olfactory cavity surrounded by mucus-producing cells. (D) 7 hPH: olfactory
cavity. Arrow: pectoral fin. Arrowhead: adhesive glands.
Figure 5 Electron micrographs of Betta splendens showing some structures during development. (A) 17 h post-hatching (hPH);
(B)23hPH;(C)85hPH;(D–F) 65 hPH: neuromasts on the lateral line region and around the eye; (G) 86 hPH: neuromasts on
the lower jaw region; (H) 936 hPH: detail showing scales in juveniles. Thick arrows: ciliated cells. Thin arrows: neuromasts.
after fertilization and zygote formation (egg cell).
This process varies greatly among vertebrates and
depends on the amount of egg yolk (Gilbert, 2003).
The eggs of B. splendens undergo meroblastic or partial
cleavage because the mitotic divisions occur only in
the animal pole of the egg. This type of cleavage
is typical of fish that accumulate a large amount
of yolk (Leme dos Santos & Azoubel, 1996; Gilbert,
2003; Takeuchi et al., 2008), such as Gymnocorymbus
ternetzi (Celik et al., 2012), F. marmoratus (Alberto

8Valentin et al.
Figure 6 Photomicrographs of Betta splendens.(A) 72 h post-hatching (hPH). (B)96hPH.(C) 156 hPH. (D) 156 hPH. Circle:
Heart. Black thick arrow: Artemia sp. ingested by the larva. Black thin arrow: mouth opened for respiration.
Table 2 Main events during larval development of B. splendens
Developmental
stage Main events
29 hpf Hatching begins. Displays total length of 2.47 ±0.044 mm, little pigmented eyes, closed mouth, adhesive
glands and the presence of melanophores in the anterior region (Fig. 2A, Fig. 3Band Fig. 4A,B)
11 hPH Eyes more pigmented, reduced yolk volume, evident notochord and the presence of melanophores in a
region of the yolk (Fig. 2B)
17hPH Total length of 2.66 ±0.068 mm and mouth opening (Fig. 5A)
49 hPH Eyes and body were more pigmented (Fig. 2D)
73hPH Yolk totally absorbed and total length of 3.20 ±0.176 mm (Fig. 2E)
432 hPH Dorsal and anal fins appear (Fig. 2F)
768 hPH Characteristics similar to adult fish and total length of 17.24 ±2.064 mm (Fig. 2G)
hpf: hours post-fertilization; hPH: hours post-hatching.
Weber et al., 2012) and Brycon gouldingi (Faustino et al.,
2010b).
The morula stage is reached once the zygote has
divided into 64 blastomeres. Through these divisions,
the number of cells increases while the volume of each
individual cell decreases (Wolpert et al., 2000; Gilbert,
2003), as observed for B. splendens. Immediately after
the morula stage, the blastula, which is characterized
by the blastoderm, is formed (Marques et al.,
2008).

Early development of B. splendens 9
The gastrulation process is characterized by epiboly
and involution movements (Wolpert et al., 2000;
Gilbert, 2003; Kunz, 2004). The epiboly movement
consists of the spreading of the blastoderm toward
the vegetal pole (Gilbert, 2003; Faustino et al., 2010a).
After the blastoderm has engulfed at least the half of
the yolk, the involution movement initiates (Gilbert,
2003; Kunz, 2004). Wolpert et al. (2000) and Kunz (2004)
show that this process leads to the formation of two
layers: the epiblast, which gives rise to the ectoderm,
and the hypoblast, which gives rise to mesoderm and
endoderm.
Temperature strongly influences the duration of
embryonic and larval development (Morrison et al.,
2001; Martell et al., 2005): the higher the temperature,
the shorter developmental time and vice versa (Leme
dos Santos & Azoubel, 1996; Martell et al., 2005).
The embryonic development of B. splendens was
slower compared with other ornamental species. The
cardinal tetra, Paracheirodon axelrodi, hatches 19–20 h
post- fertilization (hpf) at an average temperature of
26 ±1ºC (Anjos & Anjos, 2006); Gymnocorymbus ternetzi
hatches 20–21 hpf at 24 ±0.5ºC (Celik et al., 2012);
however, A. ocellatus development was even slower, as
it hatched after 46–58 hpf at a temperature of 27.5ºC
(Paes et al., 2011). This variability can be explained
by factors such as temperature and interspecific
variation.
Fish species can be classified either as precocial or
altricial depending upon the strategy adopted. The
larvae of precocial species hatch from the eggs in the
juvenile stage, while the altricial species hatch before
this stage and the larvae exhibits an undifferentiated
developmental stage (Bejarano-Escobar et al., 2010).
Betta splendens is an altricial species, as it hatches with
several organs and systems in differentiation.
The newly hatched B. splendens larvae had an
average TL of 2.47 ±0.044 mm, higher than other
sedentary species such as F. marmoratus (Alberto Weber
et al., 2012) that hatches at 1.27 ±0.4 mm and G. ternetzi
(Celik et al., 2012) at 1.44 mm, but smaller than P.
axelrodi (Anjos & Anjos, 2006) which hatches at 2.9 ±
0.2 mm. Coleman & Galvani (1998) stated that there
is a relationship between egg size and the length of
newly hatched larvae and analysed it in a wide range
of tropical species, concluding that the larger the egg
size, the longer would be the newly hatched larvae.
However, there are few studies available on this topic.
After hatching, B. splendens larvae have adhesive
glands that consist of mucous cells present in the head.
These glands enable the larvae to remain attached
to the nest, to increase parental efficiency (Araújo-
Lima & Bittencourt, 2001). These glands are similar
to those described for A. ocellatus (Paes et al., 2011),
Cichlasoma dimerus (Meijide & Guerrero, 2000) and
Hoplias malabaricus (Araújo-Lima & Bittencourt, 2002).
In the current study, the mouth opened very
quickly, while depletion of the yolk sac happened
later. According to Yúfera & Darias (2007), when
the larvae initiates exogenous feeding it is important
that all structures related to ingestion, digestion and
assimilation are ready. Furthermore, it is necessary
to highlight the importance of sensory structures
such as neuromasts and eyes (Bilotta & Saszik,
2001). Along with the development of fins, these
structures are essential for sensing and chasing food.
Therefore, describing these ontogenetic events will
help improve husbandry practices by making it
possible to determine the real needs of animals at
different developmental stages.
The larval stage was completed when the larvae
reached an average TL of 17.20 mm, when their
body features are similar to adults and become
juveniles (Kendall et al., 1984). The results of the
initial developmental stages of B. splendens provide
important information for the biology, breeding and
rearing of the species as well as a basis for further
studies.
Acknowledgements
We would like to thank Drs Maria do Carmo Faria Paes
and Sheryll Corchuelo for revising the manuscript
and the ornamental fish laboratory of CAUNESP for
supplying the animals.
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