Professional Documents
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hsan C elik
1
, Pnar C elik
1
, S u kran Cirik
1
, Mert Gu rkan
2
& Sibel Hayretdag
2
1
Department of Aquaculture, Fisheries Faculty, C anakkale Onsekiz Mart University, C anakkale, Turkey
2
Department of Biology, Faculty of Science, C anakkale Onsekiz Mart University, C anakkale, Turkey
Correspondence: I
C elik, Department of Aquaculture, Fisheries Faculty, C anakkale Onsekiz Mart University, Terzioglu Campus,
17100 C anakkale, Turkey. E-mail: celik_ihsan@yahoo.com
Abstract
The embryonic and larval development of black
skirt tetra, Gymnocorymbus ternetzi, are described
under controlled laboratory conditions. In addition,
major histomorphological changes and the allomet-
ric growth patterns during larval development have
been described. The laboratory-reared broodstock,
that is 1 year of age, were spawned. Hatching
occurred 2021 h after spawning at 24 0.5C.
The cleavage was nished in 2 h and the early blas-
tula stage occurred at 2:04 hours after spawning.
The gastrulation started at 3:20 hours and 30%
epiboly was observed at 3:34 hours after spawning.
Eight-somite stage was observed at 08:33 hours.
And embryonic developmental stage was completed
at 21 h after spawning. The newly hatched larvae
were 1442 14.3 lm in mean total length (TL).
The mouth opened at 3 days after hatching (DAH).
The yolk sac had been totally absorbed and the
larvae started to swim actively within 34 days.
Notochord exion began at 11 DAH. The metamor-
phosis was completed and the larvae transformed
into juveniles at 32 DAH. In this paper, the full
developmental sequence from egg to juvenile of
G. ternetzi is described for the rst time.
Keywords: Gymnocorymbus ternetzi, embryonic
development, larval development, morphological
characteristics, allometric growth
Introduction
Characidae is a family of freshwater subtropical
and tropical sh, found in southwestern Texas,
Mexico, Central and South America and it is a
large family that comprises about 152 genera and
776 species (Nelson 1994). The black skirt tetra
(Gymnocorymbus ternetzi) is just one of many sh
in the group of tetras, Characins, is traded in the
ornamental sh industry. It is a popular species in
the trade of freshwater ornamental sh, since it is
attractive in appearance, undemanding in mainte-
nance and easily bred (Frankel 2004; Uma &
Chandran 2008). Information on embryonic and
larval development of sh is a fundamental key
which enables a closer approach to their biology
and taxonomy (Reynalte-Tataje, Zaniboni-Filho &
Esquivel 2004). Morphological features are very
important as they furnish information of life his-
tory of sh and they provide critical parameters to
hatchery production (Martinez & Bolker 2003;
Silva 2004). Early life history characters of sh
can be used in assessing phylogenetic relationships
(Richards & Leis 1984; Stiassny & Mezey 1993;
Britz 1997; Meijide & Guerrero 2000)). In addi-
tion, studies on embryonic and larval development
of any sh species can be useful in directing the
husbandry efforts of sh breeder to the specic
state and requirements of each development
stage (Marimuthu & Haniffa 2007). There is vast
literature on embryonic and larval stages of sh,
distributed among the elds of aquaculture,
applied ecology, behavioural ecology, biological
oceanography, comparative functional morphology
and physiology, sheries science, limnology and
systematic ichthyology (Takeshita, Onikura,
Matsui & Kimura 1997; Webb 1999; Arvedlund,
McCormick & Ainsworth 2000; Borges, Faria, Gil,
Goncalves & Almada 2003; Martell, Kieffer &
2011 Blackwell Publishing Ltd 1260
Aquaculture Research, 2012, 43, 12601275 doi: 10.1111/j.1365-2109.2011.02930.x
Trippel 2005; Marimuthu & Haniffa 2007; Du,
Wang, Jiang, Liu, Wang, Li & Zhang 2010). How-
ever, detailed study about the embryonic and lar-
val development of characins is scarce. In
addition, information is lacking concerning ontog-
eny of black skirt tetra from egg to juvenile. In the
present study, the embryonic and larval develop-
ment of laboratory-reared black skirt tetra (G.
ternetzi) from egg to juvenile are described in
detail for the rst time. In addition, major histo-
morphological changes and the allometric growth
patterns during larval development have been
identied.
Materials and methods
Broodstock maintenance
One-year-old black skirt tetras (G. ternetzi) were
used as broodstock in the experiment. They were
fed with commercial ornamental sh feeds (Tetr-
amin Granulat, Tetra, Germany; Protein: 46%, Oil:
12%, Fibre: 3%, Ash: 11%, Moisture: 8%), three
times a day. During broodstock culture, water
temperature, pH and conductivity were monitored
daily at 24 0.5C, between 6.0 and 6.5 and
between 100 and 200 lS respectively. Water
temperature was controlled with additional sub-
merged heaters. The photoperiod was maintained
at 9L/15D by uorescent lighting (lights on;
07:0018:00 hours). Broodstocks were kept in
40 L glass aquariums. Three pairs (3 males/3
females) were randomly selected from broodstock
tank and were placed into a 15 L spawning tank
late in the afternoon. Spawning was observed the
next day around dawn and lasted 13 h. Eggs and
larvae were obtained from three pairs of brood-
stocks.
Observations and measurements of embryos and
larvae
Fertilized eggs were collected immediately after
spawning and maintained at 24 0.5C. Some of
them were transferred into a beaker (500 mL) for
embryonic development observations. The others
were maintained in 15 L aquaria at 24 0.5C.
Eggs were observed from spawning to hatching
under an Olympus BX51 research microscope
(Hatagaya, Shibuya-ki, Tokyo, Japan) and photo-
graphed using a colour video camera (Q Imaging,
Micropublisher 3.3 RTV, Burnaby, BC, Canada).
Embryonic development stages were identied
according to Kimmel, Ballard, Kimmel, Ullman
and Schilling (1995).
Larvae were fed once a day with Artemia sp.
(INVE Aquaculture Inc., Dendermonde, Belgium)
until the end of the experiment at 32 days after
hatching (DAH). They were randomly sampled
(n = 5) daily from hatch to 18 DAH and at 1-day
interval from 18 to 32 DAH. These specimens were
observed under an Olympus SZX7 zoom stereomi-
croscope, photographed by a colour video camera
and measured using image analysis program (Q
Capture Pro, version 5.1.1.14, Dendermonde, Can-
ada). On the other hand, they were used for obser-
vations on general morphology and for the
following morphometric measurements (mm): body
depth (BD), eye diameter (ED), head length (HL),
pre-anal length (PAL), Pre-anal myomer length
(PrAM), post-anal myomer length (PoAM), snout
length (SnL), tail length, total length (TL), trunk
length (Fig. 1) Larval developmental stages were
identied according to Kendall, A.W., Ahlstrom
and Moser (1984) and differentiated into four peri-
ods I: yolk-sac larva, II: preexion larva, III: exion
larva and IV: postexion larva.
Figure 1 Morphometric characters measured in the black skirt tetra larvae.
2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 12601275 1261
Aquaculture Research, 2012, 43, 12601275 Embryonic and larval development of black skirt tetra I
C elik et al.
Allometric growth patterns were calculated as a
power function of TL (Fuiman 1983) with the
exponent and intercept obtained from linear regres-
sions on log-transformed data (Gisbert, Merino,
Muguet, Bush, Piedrahita & Conklin 2002). The al-
lometric equation Y = aX
b
of BD, ED, HL, PAL,
PrAM, PoAM, SnL, tail length and trunk length on
TL was estimated. Here Y is the dependent variable
(measured character), X is the independent vari-
able (TL), a is the intercept and b is the growth
coefcient. When isometric growth occurred,
b = 1, a positive allometric growth occurred when
b > 1 and a negative one when b < 1.
Histological observations
For histological evaluations, larvae were randomly
collected (n = 10) daily from hatch (0 DAH) to 10
and every 2 days from 10 to 32 DAH. These speci-
mens were xed in Bouins solution and 70% alco-
hol, dehydrated through a series of alcohol
concentrations, cleared in xylene and embedded in
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j)
(k) (l)
(m) (n)
(o)
(p) (r) (s)
Figure 2 The stages of embryonic development Gymnocorymbus ternetzi: (a) 2-blastomere stage; (b) 4-blastomere
stage; (c) 8-blastomere stage; (d) 16-blastomere stage; (e) 32-blastomere stage; (f) early blastula stage; (g) late blas-
tula stage; (h) early gastrula stage; (i) 30% epiboly; (j) 50% epiboly; (k) 75% epiboly; (l) 8-somite stage; (m) 11-
somite stage; (n) 13-somite stage; (o) otic capsule; (p) muscular effect; (q) otolith appearance; (r) hatching, 3 h after
hatching. Developmental stages were determined by comparison with standard zebrash embryonic stages as
described by Kimmel et al. (1995). Scale bars, ar: 500 micron, s: 1mm.
2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 12601275 1262
Embryonic and larval development of black skirt tetra I
C elik et al.
started at 13:15 hours and embryo began to spin
at 15:30 hours after spawning (Fig. 2o and p).
The eye development and brain differentiation
(forebrain, midbrain and hindbrain) has taken
place (Fig. 2q). Hatching rates were 8590% in
aquarium at 20 h after spawning. The embryonic
development completed at 21 h (Table 1).
(a)
(b)
(c)
(d)
(e)
(f)
Figure 3 Larval development of Gymnocorymbus terne-
tzi. (a) Post-hatching stage (7 h); (b) yolk-sac stage (1
DAH); (c) yolk-sac stage, the gas bladder was formed but
not completely lled (2 DAH); (d) opened-mouth stage
(3 DAH); (e) preexion larva, exogenous feeding (5
DAH); (f) preexion larva (7 DAH). Scale bars = 1 mm.
(a)
(b)
(c)
(d)
(e)
(f)
Figure 4 Larval development of Gymnocorymbus terne-
tzi. (a) Flexion stage, Notochord exion started (11
DAH); (b) exion stage, the notochord was completely
exed (12 DAH); (c) postexion larva, swim bladder
with two chambers was visible (17 DAH); (d) postex-
ion larva (19 DAH); (e) postexion larva (26 DAH); (f)
end of metamorphosis (30 DAH). Scale bars (g. a, b,
c, d, e) = 1 mm; Scale bar (g. f) = 500lm.
2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 12601275 1264
Embryonic and larval development of black skirt tetra I
C elik et al.
the larvae started to swim actively at 34 days.
The larvae started to feed exogenously within
3 days at 24 1C. The eyes were pigmented.
The larvae have a one-chambered swim bladder.
The notochord end is not exed (Fig. 3d).
57 DAH (TL: 4.29 0.074.37 0.05 mm).
The eyes became very prominent and were fully
pigmented (Fig. 3e). Second ination of swim
bladder did not occur. It formed as a single cham-
ber, increased in size and extended posteriorly. The
larvae have still primordial ns (Fig. 3e and f).
The notochord end was not exed (Fig. 3e and f).
The larvae could swim very well. Pigmentation
has increased on the head and lateral parts of the
body, black pigments were dominant, but yellow
pigments were also present (Fig. 3f).
1112 DAH (TL: 4.97 0.155.78 0.23 mm).
Primordial n was still present (Fig. 4a). Pectoral
ns were well developed. Dorsal and anal ns have
begun early differentiation (Fig. 4a and b). The
caudal-n rays begin to form. At 11 DAH, the
notochord end was slightly exed but exion is
more obvious at 12 DAH. Swim bladder increased
in size and extended posteriorly (Fig. 4b). The lar-
vae could swim very well. There were clusters of
pigment over the body but pigmentation was more
concentrated on head region (Fig. 4b).
1517 DAH (TL: 5.75 0.166.09 0.27 mm).
Second ination of swim bladder occurred between
15 DAH and 17 DAH. Swim bladder with two
chambers completely lled (Fig. 4c). Anal and dor-
sal ns begin to develop but have no rays but cau-
dal n rays were more developed (Fig. 4c). Body
shape and pigmentation pattern were not similar
to the adult sh. The stomach of larvae contained
food, ventral region of larvae was swollen and
orange.
1926 DAH (TL: 7.83 0.8211.67 1.37 mm).
At 1922 DAH, dorsal and anal ns have differen-
tiated (Fig. 4d and e). Adipose n began to form
between the dorsal and caudal ns at 22 and 23
DAH. Pigmentation has increased over the body
but the ventral trunk region was still translucent
and food particles (Artemia) could be seen in the
digestive tract. The adipose n was more obvious
at 25 and 26 DAH (Fig. 4e). Dorsal and anal ns
were more developed with the separated rays and
caudal n was forked (Fig. 4e). Body depth was
more than triple that of the previous stage and the
body shape of larvae has approached an adult
shape.
3032 DAH (TL: 14.67 0.9220.40 1.30 mm).
The body shape of larvae and pigmentation pat-
tern were similar to those of the adult (Fig. 4f).
The body was almost completely covered with pig-
ment (Fig. 4f). Larvae have a dark grey to silver
body with black vertical bars but black and grey
pigments were dominant. Two characteristic black
vertical bars were visible on posterior side of the
gills (Fig. 4f). Morphological metamorphosis was
completed and the larvae had completely trans-
formed into juveniles. The main events during
larval development of black skirt tetra are summa-
rized in Fig. 5.
Figure 7 Growth coefcients of head, trunk and tail
length during larval development stage. Each graph
represents the growth coefcients during a total length
(TL) interval.
2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 12601275 1266
Embryonic and larval development of black skirt tetra I
C elik et al.
Allometric growth
Growth of the black skirt tetra larvae followed an
exponential curve during the larval stages and is
represented by the equation y = 1.79e
0.051x
(R
2
= 0.97, n = 112) where y is total length (TL)
mm and x is DAH (Fig. 6). Four larval development
stages were observed after hatching; yolk-sac
larvae, preexion larvae, exion larvae and postex-
ion larvae. The yolk sac has been completely con-
sumed at 4 DAH, when TL was 4.03 0.14 mm.
Notochord has been exed between 10 DAH and
12 DAH, at 5.34 0.57 mm. All the meristic char-
acters were completely developed and juvenile stage
(a)
(b)
(c)
Figure 10 Sagittal sections of black skirt tetra larvae.
(a) 2 DAH (Olympus BX51 1009), (b) 3 DAH (Olym-
pus BX51 409), (c) 4 DAH (Olympus BX51 409). at,
alimentary tract; e, eye; ga, gill arches; l, liver; n, noto-
chord; oe, oesophagus; ph, pharynx; s, stomach; sb,
swim bladder; t, teeth; ys, yolksac.
(a)
(b)
(c)
Figure 11 Sagittal sections of black skirt tetra larvae.
(a) 16 DAH (Olympus SZX7 zoom stereo microscope
209), (b) 24 DAH (Olympus SZX7 zoom stereo micro-
scope 12.5x), (c) 32 DAH (Olympus SZX7 zoom stereo
microscope 89). df, dorsal n rays; e, eye; g, gill; ga,
gill arches; gl, gill lamellae; i, intestine; l, liver; m,
mouth; n, notochord; oe, oesophagus; ph, pharynx; s,
stomach; sb, swim bladder; sb1, rst chamber of swim
bladder; sb2, second chamber of swim bladder; t, teeth.
2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 12601275 1268
Embryonic and larval development of black skirt tetra I
C elik et al.
(Kimmel et al. 1995), 1.47 0.20 mm for Corydo-
ras aeneus (Callichthyidae) (Huysentruyt & Adria-
ens 2005), 1.799 0.0214 mm for Corydoras
paleatus (Callichthyidae) (U
C elik et al.
those of other characin (Romagosa, Narahara &
Fenerich-Verani 2001; dos Anjos & dos Anjos
2006; Pan, Zhan & Gong 2008; Faustino, Nakaghi
& Neumann 2010) and zebrash Danio (Brachyda-
nio) rerio (Kimmel et al. 1995). Egg hatching time
of black skirt tetra is different from that of others.
Early larval development of G.ternetzi was divided
into four different periods: Yolk-sac larva; the pres-
ence of a yolk sac ventrally in the body, between
hatching and 4 DAH. Yolk sac was absorbed and
larvae swim actively 34 days after hatching and
the onset of exogenous feeding occurred 3 days
later. Preexion larva: this period begins at absorp-
tion of yolk sac and ends at the start of upward
exion of the notochord (between 4 and 11 DAH).
Flexion larva: this period (the period during noto-
chord exion) is characterized with the hypural
bones assuming a vertical position, between 11
and 12 DAH. Postexion larva: the period between
completion of exion and the juvenile stage,
1232 DAH. Our ndings may provide a basis for
further studies to determine the complete early life
history of black skirt tetra G. ternetzi and detailed
studies such as these may be helpful for commer-
cial production of some ornamental sh. The
results of this study can contribute to a better
understanding of the embryonic and larval devel-
opment of other commercial characin larvae. They
can be used to explain some aspects of the early
life history at culture conditions and can help to
develop better larval culture methodologies in
hatchery. Similarly, they will be helpful to increase
success rates in the larval culture of some freshwa-
ter ornamental sh species. New approaches can
be brought to solve many problems in larval cul-
ture of many other species.
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