Zygote 18 (May), pp. 131–144.


C Cambridge University Press 2009

doi:10.1017/S096719940999013X First Published Online 27 October 2009

Development of the neotropical catfish Rhamdia quelen

(Siluriformes, Heptapteridae) incubated in different
temperature regimes

2 2 2 2
Alana Marielle Rodrigues-Galdino , Camila Valente Maiolino , Mariana Forgati , Lucélia Donatti ,
3 4 1,2
Jorge Daniel Mikos , Paulo César Falanghe Carneiro and Flavia Sant’Anna Rios
Universidade Federal do Paraná, Departamento de Biologia Celular, Curitiba; Pontifı́cia Universidade Católica do Paraná,
Setor de Piscicultura (LAPEP), São José dos Pinhais; and Empresa Brasileira de Pesquisa Agropecuária
(EMBRAPA)–Tabuleiros Costeiros, Aracaju, Brazil

Date submitted: 23.02.2009. Date accepted: 04.06.2009

Summary
The developmental stages for the embryonic and larval periods of the silver catfish (Rhamdia quelen)
kept at different temperatures (21, 24, 27 and 30◦ C) are described. Fish were analysed under light and
scanning electron microscopy. For embryonic development, we described 25 stages, which were grouped
into seven periods named zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatching
periods. For larval development, we defined three stages (early, mid, and late larvae). Additionally,
the main ontogenetic events during the post-larvae and early juvenile periods were also described. This
species presents a well developped lateral line and chemosensory systems that grow up during the larval
period, maturing in the post-larvae. All tested temperatures are viable to R. quelen development, but a
shorter incubation period was necessary to complete the development at lower temperatures. However,
some malformations (heart edema) were verified at 30◦ C.

Keywords: Developmental biology, Embryology, Fish, Jundiá, Organogenesis

Introduction Rapid growth rates are typical of the fish early

The description of embryonic stages of a fish species
could allow taxonomic identification of embryos that
are in the natural environments. This knowledge is
important in ecological and conservational studies,
as sites of spawning could be correctly recognized
(Alves & Moura, 1992). Moreover, the larval production
may be optimized using data concerning the best
environmental conditions for incubation (Alves &
Moura, 1992).
1
All correspondence to: Flavia S. Rios. Universidade
Federal do Paraná, Departamento de Biologia Celular, PO
Box 19031, CEP 81531–990, Curitiba–PR, Brazil. e-mail:
flaviasrios@ufpr.br
2
Universidade Federal do Paraná, Departamento de Biologia
Celular, PO Box 19031, CEP 81531–990, Curitiba–PR, Brazil.
3
Pontifı́cia Universidade Católica do Paraná, Setor de
Piscicultura (LAPEP), R. Benjamim Claudino Barbosa n◦ 3184,
CEP 83005–970 – São José dos Pinhais, PR, Brazil.
4
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)–
Tabuleiros Costeiros, PO Box 44, CEP 49025–040, Aracaju, SE,
Brazil.
life history. The optimum conditions to embryonic
and larval development vary among the fish species.
Temperature is a critical factor in determining growth
rate, affecting the developmental timing, size at
hatching, efficiency of yolk utilization and formation
and function of key tissues and structures (Kamler,
1992; Saka et al., 2004; Gabillard et al., 2005). High
temperatures may have teratogenic effects or cause
long-term changes in muscle cellularity (Finn, 2007).
The silver catfish (Rhamdia quelen) is a eurythermal
fish species native to South and Central America that
tolerates temperatures from 15 to 34◦ C (Chippari-
Gomes et al., 1999) and survives during cold winters,
growing faster in the warm summer (Barcellos et al.,
2002). Studies carried out in the last years with
this species have pointed out its great potential for
aquaculture (Carneiro et al., 2003). Consequently, it
has been of great interest among researchers and fish
culturists in Brazil.
The early development of R. quelen has been
described only briefly (Godinho et al., 1978; Pereira
et al., 2006), and a developmental staging series for this
132 Rodrigues-Galdino et al.
species remains incomplete. A staging series is a useful
standardization tool that provides accuracy in devel-
opmental studies (Fujumura & Okada, 2007). Develop-
mental staging allows us to compare the developmental
processes between the model species and related spe-
cies to reveal underlying mechanisms of evolutionary
changes among them (Fujimura & Okada, 2007).
The commercial production of Siluriformes (catfish)
has grown in the world; however, the study of
developmental biology of Brazilian catfish is incipient
(Godinho et al., 1978; Luz e Zaniboni Filho, 2002;
Luz et al., 2001; Pereira et al., 2006). The mean water
temperature in South Brazil, where the most extensive
culture of silver catfish exists, ranges between 15
and 30◦ C (Chippari-Gomes et al., 1999). According
to Gomes et al. (2000), studies concerning physico-
chemical parameters of the water and growth in
captivity are essential to improve the use of this species
in fish culture.
The present study investigated the embryonic-
larval development of R. quelen incubated in different
temperatures and proposed a staging system to this
species by morphological criteria.
Materials and methods
Experimental design
Mature males and females of R. quelen were induced
to reproduce by hypophysation. Six replicate chambers
were used for each of four constant temperatures: 21◦ C,
24◦ C, 27◦ C and 30◦ C. Each chamber consisted of a
1.3 l flowthrough incubator and each set of chambers
was maintained in a 100 l tank. Dissolved oxygen
was monitored by an YSI–55 (Hexis) oximeter and
kept between 6 mg/l and 7 mg/l by aeration. The
eggs were gradually adapted to each test temperature.
Approximately 3000 fertilized eggs were placed into
each chamber.
Sampling and staging
Embryos from each temperature were sampled every
30 min during the first 19 h, and thereafter in intervals
of 1 h until all viable eggs at each temperature
had hatched. Larvae were sampled every 12 h
until complete yolk absorption. Samples of embryos
were pipetted carefully from each chamber and
photomicrographs of live embryos were acquired
with a Sony Cyber Shot camera on a Quimis light
microscope. Developmental stages were determined
using criteria set out in Kimmel et al. (1995). Embryos
were not dechorionated and descriptions were based
on intact individuals. Age was recorded in hours post
fertilization (hpf) or days post fertilization (dpf). At the
end of larval period, the post-larvae were transferred
to external concrete tanks and fed on zooplankton.
Samples of post-larvae with 7 and 21 days were fixed
and processed to light and electron microscopy.
Light microscopy
Embryos and larvae fixed in 4% paraformaldehyde
(PFA) in 0.1 M PBS were rinsed in PBS buffer,
dehydrated in a graded ethanol series, and embedded
in Leica
R
historesin. Samples fixed in ALFAC were
embedded Paraplast Plus R
after dehydration. Sections
were stained with haematoxylin–eosin or toluidine
blue and analysed in a Quimis R
light microscope.
Scanning electron microscopy
After fixation in Karnovski medium (2% paraformalde-
hyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer, pH 7.2 at 4◦ C), embryos/larvae were rinsed in
buffer, dechorionated manually using sharp forceps,
and dehydrated in a graded ethanol series. Samples
were critical-point dried in liquid CO2 in a BalzersR

R
CPD–010, coated with gold in a Balzers SCD–030, and
analysed in a JEOL R
-JSM 6360 LV scanning electron
microscope.
Results
Embryonic development of R. quelen began with
fertilization and ended with hatching out from
the chorion (stages 1–25; Table 1). The embryonic
development was subdivided into seven periods:
zygote, cleavage, blastula, gastrula, segmentation,
pharyngula, and hatching. The larval development was
subdivided into three periods: early larval, mid larval,
and late larval (stages 26–28), and is characterized by
the presence of the yolk vesicle. Subsequently, after
exhaustion of the yolk, the organisms were considered
post-larvae (stage 29).
Rhamdia quelen embryos presented a negative
relationship (r2 = 0.9951) between time of development
and incubation temperature (Fig. 1A, Table 1).
Therefore, at 30◦ C, larvae hatched after 19 h of
incubation, while they spent 43 h to hatch at 21◦ C.
The duration of each period varied according to each
test temperature. At 21◦ C, the time of segmentation
and pharyngeal periods was much higher than that at
higher temperatures (Fig. 1B).
The Table 1 summarizes the main developmental
landmarks for R. quelen. The major morphological
features in each stage were standardized in spite of
temperature, as described below.
 Stages of R quelen development 133

Table 1 Periods and stages of development of Rhamdia quelen incubated at 21, 24, 27 and 30◦ C.

Hours post fertilization (hpf)

Period Stage 21◦ C 24◦ C 27◦ C 30◦ C Description

Embryo
Zygote 1 0:00 0:00 0:00 0:00 1 cell – cytoplasm streams toward animal pole to form the blastodisc
Cleavage 2 0:40 0:40 na na 2 cells – partial cleavage
3 1:00 1:00 1:00 na 4 cells – 2 × 2 array of blastomeres
4 1:30 1:15 1:10 1:00 8 cells – 2 × 4 array of blastomeres
5 2:00 1:30 1:20 1:10 16 cells – 4 × 4 array of blastomeres
6 2:20 1:40 1:30 1:20 32 cells – 4 × 8 array of blastomeres, cells starts to compact
7 2:40 1:50 1:45 1:30 64 cells – 2 layers of 32 blastomeres, YSL forms
Blastula 8 3:00 2:00 2:00 1:45 Early blastula – 128 to 256 blastomeres, forming a half sphere
9 4:00 3:00 2:30 2:00 Mid blastula – 512 blastomeres or more
10 5:00 4:00 3:00 2:30 Late blastula – blastodisc flattening
11 5:30 5:00 4:45 4:00 30% Epiboly – blastoderm highly flattened with uniform thickness;
margin reaches 30% of distance between the animal and vegetal
poles
Gastrula 12 6:00 6:00 5:15 5:00 50% Epiboly – margins of the blastoderm reaches 50% of distance
between the animal and vegetal poles; germ ring and, after,
embryonic shield is visible
75% Epiboly – blastoderm covers 75–80% of the embryo; dorsal side
is thicker
13 9:30 6:30 6:00 5:30 Yolk plug – yolk plug in the vegetal pole
14 10:00 7:30 7:00 6:00 Neurula – blastoderm fully covers the yolk plug closure; neural
groove visible; embryo become elliptical
15 11:00 9:00 8:00 7:00
Segmentation 16 11:30 9:30 8:30 7:30 2–3 Somites – the first pairs of somites and optic primordium are
visible
17 15:00 11:15 9:00 8:00 6 Somites – brain neuromeres, optic vesicles, nostrils (primordial
olfactory organ), and notochord visible
18 15:30 12:00 10:00 8:30 10 Somites – Kupfer’s vesicle and otic placode formation. Optic
vesicle more evident
14 Somites – mesencephalon slightly prominent, optic lens, YE
barely forming
19 16:30 13:00 11:00 9:00 18 Somites – otic vesicle, at least 3 hindbrain neuromeres, tail bud
begins to protrude, first muscular twitches
20 17:30 14:00 11:30 10:30 25 Somites – V-shaped trunk somites, side-to-side flexures, tail and
YE well extended, otoliths in the otic vesicle, dorsal aorta visible,
Kupfer’s vesicle disappeared
21 20:30 15:30 15:00 12:30
Pharyngula 22 26:00 17:00 16:00 14:30 Early pharyngula – ∼30 myomeres, intense motility (embryo rotate
inside the chorion), foregut development (but the mouth and the
anus remained closed), pericardial cavity visible, mesencephalon
divided in two hemispheres
23 40:00 19:00 18:00 16:00 Mid pharyngula – heartbeat (65–69 bpm), weak circulation, midgut
visible, YE disappeared, embryonic fin (dorsal continuous with
caudal and anal)
24 41:00 21:00 19:00 18:00 Late pharyngula – strong afferent and efferent circulation
(∼80 bpm), ∼40 myomeres, embryos folded (1/2 trunk) inside the
chorion; chorion deforms with embryo movements, gaps
correspondent to the mouth and operculum are visible, midgut
visible
Hatching 25 43:00 26:00 20:00 19:00 Hatching – low motility just before hatching, ∼100 bpm, no
pigmentation
Larvae 26 na 36:00 na na Early larva – eye became pigmented, the mouth is a elliptical gap,
barbells buds (3 pairs), anal pore and opercula open, pharyngeal
pouches start to develop, neuromasts present in trunk and
cephalic region
134 Rodrigues-Galdino et al.

Table 1 (Cont.)

Hours post fertilization (hpf)

Period Stage 21◦ C 24◦ C 27◦ C 30◦ C Description

27 na 66:00 na na Middle larva – body became pigmented, longer barbells with small
and numerous protuberances (primordial taste buds), maxillary
barbells larger than mental barbells, inferior and superior lips
become defined
28 na 90:00 na na Late larvae – barbells well developed, taste buds on barbells and
lips, protruded jaws, deep nostrils with ciliated cells, yolk sac very
reduced, swim bladder inflated, and beginning of exogenous
feeding
Post-larvae 29 na 7 dpf na na Yolk sac absent, barbells and fins well developed
Juvenile 30 na 21 dpf na na Buccal and pharyngeal teethes, presence of large thymus, lateral
line pores
hpf = hours postfertilization; dpf = days postfertilization YE = yolk extension, YSL = yolk syncytial layer; na = not analysed.

Embryonic development Period 4 – gastrula

Period 1 – zygote
A distinct perivitelline space emerges around the
fertilized egg, as the chorion lifts away from the
cortex of the yolk sphere. The cytoplasm streams and
accumulates on the animal pole, forming the active
cytoplasm (blastodisc) (Fig. 2).
Period 2 – cleavage
The cleavage period consisted of six stages (stages
2–7; Fig. 3). This period was characterized by a
series of mitotic divisions on the active cytoplasm
(meroblastic cleavages) that resulted in 64 blastomeres.
After each cleavage the blastomeres decrease in size.
During cleavage period, embryos formed in average
400 blastomeres per hour at 30◦ C. At 21◦ C, this rate
decreased to half (200 cells per hour; Fig. 4A).
Period 3 – blastula
The blastula period consisted of four stages (stages
8–11). The early blastula (stage 8) was characterized
by a blastoderm with 128 or 256 cells, forming a half
sphere elevated from the yolk cell. The mid-blastula
(stage 9) has 512 or few thousands of blastomeres, an
outer enveloping layer (EVL) and a peripheral yolk
syncytial layer (YSL). The YSL is a syncytial layer
located in the intersection of yolk and blastoderm that is
perceptible in live embryos under light microscope as a
darker region. The edges of the blastoderm are jagged
(Fig. 5A, B). During the late-blastula stage (stage 10),
the blastoderm flattens due blastomeres intercalation,
forming a compact uniform mass (Fig. 5C). At the end
of this period, the blastoderm was highly flattened and
starts the morphogenetic movement known as epiboly.
During the stage 11, the margins of blastoderm cover
30% of the yolk cell (Fig. 5D).
The gastrula period consisted of four stages (stages 12–
15). The stage 12 was characterized by 50% (Fig. 6A)
of epiboly and the presence of the germ ring around the
margin of the blastoderm (Fig. 6C, D). The germ ring is
formed by the epiblast and hypoblast superposition.
Subsequently the embryonic shield is formed by
thickening of the germ ring in the future dorsal side
of the embryo. The shield extends from the germ
ring toward the animal pole. As epiboly proceeds, the
blastoderm gradually covers the whole yolk cell (Fig.
6E–H). During the neurula stage (stage 15, Fig. 6I, J)
the blastoderm fully covers the yolk plug, closing the
blastopore; the neural groove is visible; and the embryo
became elliptical. The gastrulation, easily visible by per
cent of epiboly, lasted about the half of time in higher
temperatures (Fig. 4B).
Period 5 – segmentation
The segmentation period consisted of six stages
(stages 16–21; Fig. 7). Somite formation, beginning
of organogenesis, and compartmentalization of the
brain characterized this period. The mesodermal
segmentation begins to form the somites. Together
to the development the three first somites, the optic
vesicles become conspicuous. The Kupfer’s vesicle
appears and disappears during this period. The tail
bud starts to protrude and the yolk extension (YE) is
formed. The first muscular twitches occur in the stage
20 (18 somites). The somites develop into V-shaped
myotomes (Fig. 8) that allow side-to-side flexures of
the embryo body in the stage 21 (25 somites).
Period 6 – pharyngula
The pharyngula period consisted of three stages (stages
22–24; Fig. 9). The early-pharyngula embryos (stage
22) have about 30 myotomes and present intense
 Stages of R quelen development 135

Figure 1 Effect of incubation temperature on time of hatching for Rhamdia quelen. (A) Age at hatching (hpf; hours post-
fertilization) of the embryos incubated in different regimes of temperatures. Mean times (hpf) were fitted to a polynomial
function y = 0.4444x2 − 25 267x + 377.3, where y represents time of hatching (hpf) and x represents the temperature (◦ C).
(B) Duration of each period (from cleavage to hatching) in function of the time (hpf) of the embryos of Rhamdia quelen incubated
at four different temperatures.

motility (Fig. 9A). During this period, the embryo body
elongated and the late-pharyngula embryos (stage 24)
present about 40 myotome, needing to fold (1/2 trunk)
inside the chorion. The blood circulation begins during
this period. The weak circulation visible in the stage 23
became strong in the stage 24. The pericardial cavity is
visible in the stage 22 and the first heartbeats occur in
the mid-pharyngula (stage 23). Remarkable structures
such as primordial fins (embryonic fin), midgut, and
gaps related to mouth and operculum became visible
in the pharyngula period. A pair of otoliths also appears
as small granules lying against the inner surface of each
otocyst. Blood circulates in all three vitelline veins. The
pharyngula period presented greater variation among
temperatures, lasting 17 h at 21◦ C and only 4.5 h at 30◦ C
(Table 1).
136 Rodrigues-Galdino et al.

Figure 2 Zygote period of Rhamdia quelen. (A, B) Just fertilized.

(C, D) The cytoplasm streams and accumulates on the animal
pole, forming blastodisc (stage 1). (A) and (C) are light
microscope images, and (B) and (C) are scanning electron
microscopy images. Scale: 500 μm.

Figure 3 Cleavage period of Rhamdia quelen. Embryos fixed in Figure 4 Development of Rhamdia quelen embryos incubated
4% paraformaldehyde (light microscopy) with: (A) two (stage in four different temperatures (21, 24, 27, and 30◦ C) as a
2), (B) four (stage 3), (C) eight (stage 4), and (D) 16 blastomeres function of the time (hpf, hour post-fertilization). (A) Number
(stage 5). Scale: 500 μm. of blastomeres. (B) Per cent of epiboly. (C) Number of somites.

Period 7 – hatching
The hatching period consisted of one stage (stage 25).
The hatching period was characterized by low motility
just before hatching. The heart reaches 100 bpm.
The hatch larvae do not present any pigmentation.
Developmental time was inversely proportional to
temperature (Fig. 1A). At 30◦ C, larvae hatched after
19 h of incubation, while they lasted in average 43 h to
hatch at 21◦ C (Fig. 1B, Table 1).
 Stages of R quelen development 137

Figure 5 Blastula period of Rhamdia quelen. Life embryos

under light microscope. (A) Early blastula (stage 8); (B) Mid
blastula (stage 9); (C) Late blastula (stage 10); and (D) 30%
epiboly (stage 11). Scale: 200 μm.

Larval development
Period 8 – larval
The larval period was divided into three stages
(stages 26–28; Fig. 10). Larval development began after
the hatching and finished with the complete yolk
absorption. The yolk cell is much reduced in stage 28.
The eyes became pigmented in the early-larvae (stage
26), but body pigmentation comes into view in the mid-
larvae (stage 27). The mouth is an elliptical gap and
three pairs of barbells buds grow around it in the early
larvae. Inferior and superior lips develop and the jaw
became protruded in the mid-larvae. Between stages 27
and 28, the larvae inflate the swim bladder and initiate
exogenous feeding.

Organogenesis
Nervous/sensory systems
Neural tube forms at the end of gastrulation (stage 15).
At the stage 17, the first neuromeres and the buds of
some important sensorial structures, as optic vesicles
and nostrils are visible (Fig. 11). The otic vesicles
(Fig. 12A) appear later, during stage 18. The sensorial
epithelium (macula) is located in anterior-ventral of
the internal face of the otic cups and the otoliths
were observed inside them in stage 21. Forebrain,
midbrain and at least three hindbrain neuromeres
are clearly visible in stage 20 (Fig. 11F). The early
pharyngula (stage 22) presents the forebrain divided in
Figure 6 Blastula (late blastula) and gastrula periods of
Rhamdia quelen. (A) 50% epiboly (stage 12); (B) late blastula
(stage 10); (C) 50% epiboly with germ ring formation (stage
12); (D) 50% epiboly (stage 12); (E, F) 75% epiboly (stage
13); (G, H) yolk plug (90% epiboly; stage 14); and (I, J)
neurula stages (stage 15). Embryos of the left column were life
(light microscopy), while embryos of the right column were
analysed under scanning electron microscope. All embryos
are in lateral view, except to (H), that is in view from vegetal
pole. Scale: 200 μm.
138 Rodrigues-Galdino et al.

Figure 7 Segmentation period of Rhamdia quelen. Life embryos with: (A) 2–3 somites (stage 16); (B) six somites (stage 17);
(C) 10 somites (stage 18); (D) 14 somites (stage 19); (E) 18 somites (stage 20); (F) 25 somites (stage 21). Symbols: e = eye, k =
Kupfer’s vesicle, m = myomere, s = somite, y = yolk, ye = yolk extension. Scale: 200 μm.

Figure 8 Muscle formation of Rhamdia quelen. (A) Life embryos with 10 somites (stage 18); and (B) 25 somites (stage 21).
(C) Histological sagittal section from muscle trunk of just hatching larvae. Symbols: m = myomere, s = somite, y = yolk, ye =
yolk extension. Scale: (A, B) 100 μm; (C) 10 μm.

two hemispheres. The eyes develop gradually, became
pigmented at the early larval period (stage 26). The
lateral line system starts to form before hatching, when
the first neuromasts are visible (Fig. 12A). A complex
set of pores lateral line system is present in the trunk
and cephalic region of the juvenile. The nostrils of
the larvae became progressively deeper and covered
by ciliated cells (Figs. 11A–E and 12C). The early
larvae present nostrils positioned laterally, but these
structures displace to a terminal position as the jaw
develops. The otic cups (Figs. 11F, 12D) also move
to an anterior position. The barbells develop after
hatching (Figs. 10 and 11). Three pairs of barbells buds
develop around the mouth, being one on maxilla and
two mental barbells. In stage 27, small and numerous
protuberances (primordial taste buds) appear on the
 Stages of R quelen development
Figure 9 Pharyngula period of Rhamdia quelen. Life embryos in stages: (A) Early pharyngula (stage 22); (B) mid pharyngula
(stage 23); (C) late pharyngula (stage 24). Symbols: h = heart (pericardial cavity), o = otic cup, y = yolk, ye = yolk extension.
Scale: 500 μm.
Figure 10 Larval period of Rhamdia quelen. Scanning electron
microscopy of: (A, B) early larvae (stage 26, being A younger
than B); (C) mid larvae (stage 27); (D) late larvae (stage 28).
Symbols: a = anal pore, bf = buccopharyngeal cavity (oral
slit), f = embryonic fin, nt = nostril, op = opercular cavity,
y = yolk, head arrows = barbels. Scale: 500 μm.
barbells buds (Fig. 12E, F). During the late-larvae
stage (stage 28) the barbells elongated, turn into well
developed structures, and numerous taste buds cover
the barbells and lips (Fig. 12G).
Digestive system
During the embryonic and the major part of larval
periods, the development depends on the endogenous
nutrition (yolk). The YSL is visible in the mid blastula
(stage 9). The yolk cell decrease gradually, being
139
completely consumed at the end of the larval period.
The foregut is visible at the stage 22, while midgut is
distinct at the stage 23. Although, the larvae hatched
with an opened mouth (represented by an elliptical slit
positioned ventrally in the cephalic region), the anal
pore open only after hatching (stage 26). However, the
larvae can consume exogenous food even before the
yolk exhaustion. The mid larvae (stage 27) have distinct
superior and inferior lips that became covered with
taste buds in the stage 28. Both taste buds and teeth are
present in post-larvae pharynx (Fig. 12H).
Cardiorespiratory system
The pericardial cavity appears in the stage 19 and
the dorsal aorta is visible at the end of segmentation
period. The first heart beats (65 bpm in average) occur
in the mid pharyngula (stage 23). In this period the
embryo presents weak circulation. Gradually, the heart
frequency increase to 100 bpm at the hatching and
the blood circulate faster. The operculum open before
hatching, but the early larvae (stage 26) present only
primordial gill arches, represented by discrete groups
of mesoderm cells on pharyngeal region. Some embryo
incubated at 30◦ C presented heart edema (Fig. 13).
Locomotory system
The first somites appear in stage 16. During
segmentation, about five pairs of somites were formed
per hour at 30◦ C against a rate of less than three somites
per hour at 21◦ C (Fig. 4C). The first muscle contraction
occurs during the stage 20, when the embryos have
about 18 pair of somites. At the stage 21, the somites
become ‘V-shaped’ and the embryo present side-to-
side flexures. The hatching embryo presents about
40 myomeres. During the pharyngula period, the
140 Rodrigues-Galdino et al.

Figure 11 Nervous and sensory systems of Rhamdia quelen. (A) Hatching larvae (stage 25); (B) early larvae (stage 26); (C) late
larvae (stage 28); (D) post-larvae (stage 29); (E) juvenile (stage 30); (F) hatching larvae (stage 25). Symbols: d = diencephalon,
e = eye, hb = hindbrain, ht = heart (pericardial cavity), m = midbrain, n = notochord, nt = nostril, o = otic cup, op = opercula,
t = telencephalon, y = yolk, head arrows = lateral line system pores, ∗ = barbels. (A–E) Scanning electron microscopy;
(F) histological sagittal section. Scale: 500 μm.

embryos form the embryonic fin, characterized by
a dorsal fin continuous with a ventral fin (Fig. 10).
The definitive fins differentiate during the post-larval
period.
Discussion
The knowledge of the embryonic and larval stages,
as well as, the control of water temperature under
laboratory condition is of great help to optimize
time and labour, nevertheless the knowledge of the
embryonic and larval development under different
temperature regime is important to recognize the best
way to produce fingerlings of good quality.
The nomenclature and number of embryonic and
larval periods are widely divergent in literature.
The classification adopted for the present study
was based on Kimmel et al. (1995). We described
seven periods for the embryonic development (zygote,
cleavage, blastula, gastrula, segmentation, pharyngula
and hatching), and three larval periods (early larvae,
mid larvae, and late larvae). Moreover, we described
the main ontogenetic events during the post-larvae
period and the begging of the juvenile phase.
In species with predominant visual sense to food
perception, as Cyprinus carpio (Appelbaum e Riehl,
1997) and Brycon amazonicus (Neumann, 2008), the eyes
are well developed even in larvae. The R. quelen eyes
became pigmented during early larvae stage. However,
like other catfishes (Britzki et al., 1999), R. quelen
present relatively reduced eyes and well developed
mechanosensory (lateral line) and chemosensory (taste
buds and olfactory system) structures. During the
larval period, lips and three pairs of barbells develop
and are covered with numerous taste buds. Moreover,
the neuromasts, present since the late embryonic
period, develop in an abundant lateral line system
both in trunk and cephalic regions. Efficient lateral
line and chemosensory systems is essential to sustain
the R. quelen habits. This omnivorous species forages
predominantly at night, feeding mainly on benthic
preys (Guedes, 1980; Schulz & Leuchtenberger, 2006).
The embryonic fin remained during the overall larval
period. Van Snik et al. (1997) proposed that this fin fold
is related not only to locomotion, but also contributes
to increase the surface area to gas exchange through the
 Stages of R quelen development 141

Figure 12 Sensorial structures and pharyngeal teeth of Rhamdia quelen. (A) Neuromast; (B) longitudinal section of cephalic
region; (C) nostril; (D) otic cup; (E, F) barbel; (G) lips; and (H) pharyngeal teeth. Pictures of the left column are obtained by
scanning electron microscopy (Scale: 10 μm) and the pictures of the right column are histological sections (Scale: 500 μm).
Symbols: ne = neuromast, bn = brain, o = otic cup, g = gill arches, bf = buccopharyngeal cavity, nt = nostril, m = macula,
tb = taste buds, th = teeth, arrows = taste buds, head arrows = neuromasts (lateral line system).

skin. According to Neumann (2008), the embryonic fin
reabsorption marks the end of a period of drastic body
changes, when the small fish acquire an efficient gill
respiration and the definitive fins are formed, allowing
the change of the swimming pattern.
The improvements of structures related to swimming
and, mainly, to food detection suggest that R. quelen
is potentially able to take exogenous food even
before the exhaustion of yolk. Moreover, the presence
of abundant chemosensory structures in these early
periods suggests that this species is able to select food
since the first feeding. The mouth opening of larvae
of some fish species occur simultaneously with the
beginning of horizontal movements of swimming (Luz
142 Rodrigues-Galdino et al.

larvae to reaches the sites of accessible food resources.

Figure 13 Rhamdia quelen early larva incubated at: (A) 24◦ C
(with normal pericardial cavity); and (B) 30◦ C (presenting
heart edema). Symbols: e = eye, f = embryonic fin, g = gill
arches, h = heart (pericardial cavity), o = otic cup, y = yolk,
head arrows = barbels. Scale: 500 μm.
et al., 2001) and pigmentation of the eyes (Lasker et al.,
1970). The swim bladder of R. quelen inflates during the
late larvae stage, allowing best foraging movements
coincidently to yolk drop and gut maturation.
During the first days, fish larvae consume the energy
reserve from yolk. The period of endogenous feeding is
essential to maturation of digestive structures, allowing
R. quelen late larvae start exogenous feeding even
before total consumption of the yolk. Actually, the
mouth starts to open before hatching, during the late
pharyngula stage. According to Govoni et al. (1986)
the larvae gut is functional during the first exogenous
feeding, although it is structurally and physiologically
simpler than the adult gut. In the present study, the
late larvae were fed on zooplankton even before the
complete yolk exhaustion.
The yolk reserve present in late larvae is probably a
guaranty. It diminished the risk of mortality in the case
of scarce food in the environment. The beginning of
exogenous feeding is a critic period on fish life history
as 1-day-fed post-larvae present gut atrophy, having a
limited time of survival under starvation in many fish
species (Theilacker, 1978).
Water temperature is a major determining factor in
fish biology. One obvious effect of high temperatures
is an increase in the embryonic development rate
(Garside, 1966; Vernier, 1969; Ojanguren et al., 1999;
Saka et al., 2004; Cook, et al., 2005; Martell et al., 2005;
Ninhaus-Silveira et al., 2006). Likewise, we observed a
negative relationship between the time of embryonic
development and incubation temperature in R. quelen.
Table 2 presents data from literature concerning
the duration of embryonic development in a number
of teleost species. From analysis of these data it is
possible to conclude that time of hatching is not
related to the taxonomic organization of the species.

Table 2 Previous studies of teleost development: embryonic development (time of hatching) in hours postfertilization (hpf).

Temperature Time of
Species (◦ C) hatching (hpf) Authors

Order Beloniformes
Family Adrianichthyidae Oryzias latipes 25 20:00 Gonzáles-Doncel et al., 2005
Order Characiformes
Family Anostomidae Leporinus friderici 27.6 13:20 Sanches et al., 2001
Family Characidae Brycon cephalus 26 10–11:00 Romagosa et al., 2001
Colossoma macropomum 25–26 18:30 Ribeiro et al., 1994
Piaractus mesopotamicus 25–26 22:00 Ribeiro et al., 1994
Paracheirodon axelrodi 26 19–20:00 Anjos & Anjos, 2006
Astyanax bimaculatus 24–25 17:00 Sato et al., 2006
Tetragonopterus chalceus 24–25 20:00 Sato et al., 2006
Family Erythrinidae Hoplias malabaricus 24–26 44:00 Azevedo & Gomes, 1942
Family Prochilodontidae Prochilodus scrofa 24 22:00 Cavicchioli & Leonhardt, 1993
(synonymous to P. lineatus)
Prochilodus lineatus 24 22:00 Ninhaus-Silveira et al., 2006
Prochilodus lineatus 28 14:00 Ninhaus-Silveira et al., 2006
Order Cypriniformes
Family Cyprinidae Danio rerio 28 48:00 Kimmel et al., 1995
Order Perciformes
Family Cichlidae Oreochromis niloticus 28 120:00 Fujimura & Okada, 2007
Order Siluriformes
Family Auchenepteridae Parauchenipterus galeatus 27–28 64:00 Sanches et al., 1999
 Stages of R quelen development
With few exceptions, fish embryos kept under 24–
26◦ C hatch up to 24 h. Higher temperatures usually
accelerate, while lower temperatures reduce, the
duration of embryonic development. The exceptions
to this pattern are probably related to the ecological
and/or behavioural features of each species, as the
occurrence of parental care or egg adhesiveness,
allowing a slower development.
Gomes et al. (2000) showed that R. quelen fingerlings
acclimated at 31◦ C tolerate temperatures between 15
and 34◦ C. All temperatures tested in the present
study (21, 24, 27 and 30◦ C) were found to support
successful embryonic and larval development in this
species.
R. quelen hatches faster at 30◦ C compared with
the other lower temperatures. Nevertheless, some
anatomical defects, like heart edema, were more
frequent at 30◦ C. The presence of malformations,
however, were not quantified nor analysed statistically,
needing further studies. Arenzon et al. (2002) showed
similar results in Cynopoecilus melanotaenia. In this
species the larvae incubated in higher temperatures
had hatched with morphologic anomalies and
remained immovable, making impossible its survival.
Additionally, the high metabolic rates under high
temperatures lead to faster yolk consumption and may
be hazardous in natural environments, as the yolk
could be exhausted before complete maturation of the
structures related to catching and processing of the
exogenous food and/or before the post-larvae reaches
the sites with high food concentration.
Acknowledgements
We thank Dr Dorly de Freitas Buchi (UFPR, Brazil)
for kind donation of some reagents used in our
experiment (resources from project supported by
Paraná Government–SETI).
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