Ichthyol Res (2010) 57:389–397
DOI 10.1007/s10228-010-0173-3
FULL PAPER
Growth and morphological development of laboratory-reared
larvae and juveniles of the Laotioan indigenous cyprinid
Hypsibarbus malcolmi
Yuka Ogata • Shinsuke Morioka • Kosuke Sano •
Bounsong Vongvichith • Hiroki Eda •
Hisashi Kurokura • Thongkhoun Khonglaliane
Received: 21 April 2010 / Revised: 17 June 2010 / Accepted: 18 June 2010 / Published online: 5 August 2010
Ó The Ichthyological Society of Japan 2010
Abstract The morphological development, including the
pigmentation, body proportions, fins, and survival rate for
30 days after hatching, of laboratory-reared larval and
juvenile Hypsibarbus malcolmi is described. Body lengths
(BL) of larvae and juveniles were 2.0 ± 0.2 (mean ± SD)
mm at 1 h after hatching (day 0) and 9.2 ± 0.6 mm on day
16, reaching 12.1 ± 0.9 mm on day 30. Yolk volume
decreased linearly, with the yolk being completely absorbed
by day 3 in all preflexion larvae (all specimens [3.2 mm
BL). Feeding was observed on day 2 in fish which had rapidly
undergone complete yolk absorption following mouth
and anus opening on day 1, and on day 3 in all remaining
fish. Myomere numbers were 20–21 ? 11–12 = 31–33,
although they were not clearly visible in juveniles.
Y. Ogata
K. Sano
H. Kurokura
Laboratory of Global Fisheries Sciences,
Department of Global Agricultural Sciences,
The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan
S. Morioka (&)
Fisheries Division, Japan International
Research Center for Agricultural Sciences,
1-1 Owashi, Tsukuba, Ibaraki 305-8686, Japan
e-mail: moriokas@affrc.go.jp; morishinlao@yahoo.co.jp
B. Vongvichith
Aquaculture Unit, Living Aquatic Resource Research Center,
National Agriculture and Forestry Research Institute,
Khounta Village, Sikhotabong District, Vientiane, Laos
H. Eda
IC Net Ltd., 3rd floor FSK Building, 4-1 Shintoshin,
Chuo-ku, Saitama, Saitama 330-0081, Japan
T. Khonglaliane
Namxouang Aquaculture Development Center, Aquculture
Improvement and Extension Project, Vientiane, Laos
Melanophores were few on the body during days 0–2, but
increased with growth and covered the entire upper dorsal
body surface during the juvenile stage. Body proportions
tended to become constant in juveniles. Notochord flexion
began in larvae[5.2 mm BL on day 8, and was completed in
larvae [8.4 mm BL on day 14. Specimens with full fin ray
complements were initially observed on day 22 (10.4 mm
BL in juveniles). All specimens[11.5 mm BL had attained
the juvenile stage. A high survival rate of 92.7% was esti-
mated on day 30.
Keywords Hypsibarbus malcolmi
Larvae
Juveniles

Morphology
Laos
Introduction
The genus Hypsibarbus is a relatively new taxon in
Cyprinidae under a revision of Rainboth (1996a). It occurs
across the Indochinese and Malay Peninsulas, and is con-
sidered to be most closely related to the genera Barbodes
and Propuntius. In Laos, five species belonging to this
genus, i.e., Hypsibarbus lagleri, Hypsibarbus malcolmi,
Hypsibarbus pierrei, Hypsibarbus vernayi and Hypsibar-
bus wetmorei, are known to occur (Kottelat 2001). This
species, originally described by Smith (1945) as Acrosso-
cheilus malcolmi, having been occasionally misidentified
as Puntius bramodes (see Herre and Myers 1937) or Puntius
daruphani (see Bardach 1959), and subsequently trans-
ferred to Hypsibarbus malcolmi by Rainboth (1996a), is the
largest species in size among the above five, the specimens
being observed to attain 40–50 cm in standard length (SL)
and over 3,400 g in body weight (Rainboth 1996b;
Baird and Phylavanh 1999; Baird et al. 1999). This species
is distributed in the rivers of the Mekong, Chao Phraya,
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Mae Khlong, Tapi and Pahang basins in Thailand, Laos,
Cambodia, Vietnam and the Malay Peninsula (Doi 1997;
Baird and Phylavanh 1999).
Although fish diversity in the Indochinese region is rich,
and conservation is an important regional issue, the com-
mercially-driven introductions of invasive alien fishes, e.g.,
Oreochromis niloticus, Hypophthalmichthys molitrix and
Labeo rohita (Welcomme and Vidthayanon 2003; De Silva
et al. 2006), and the aquacultural development of hybrid/
alien strains (e.g., Clarias gariepinus) (Na-Nakorn et al.
2004; Senanan et al. 2004) in recent years have given rise
to great concerns regarding the potential loss of regional
biodiversity (Nguyen and De Silva 2006). In addition, rapid
growth in the human population of this region and envi-
ronmental changes (e.g., urbanization, increasing land use
for crops) are considered to cause the declines in regional
fish diversity and fishery production, including Hypsibar-
bus malcolmi (Baird et al. 1999; Sverdrup-Jensen 2002).
These situations have led to greater emphasis being placed
on the investigation of stock assessment and the aquacul-
tural development of indigenous fish species in the region.
For H. malcolmi, extensive aquaculture trials are widely
observed in the region, but information on its present status
is unclear, since the species is often regarded as sympatric
cyprinids that are morphologically similar to H. malcolmi,
such as Barbonymus gonionotus (Vidthayanon 2001). In
addition, while some biological data on the species related
to its reproduction in the wild (e.g., spawning seasons and
grounds) have been reported (Poulsen et al. 2002), no
investigation of the biology and the morphological devel-
opment of the early life stages (larvae and juveniles) of
this species has been made as yet, even though such
information is essential for species identification and
technical improvements in seed productivity. Knowledge
of recruitment success has thus far also been limited.
Moreover, descriptions of larval and juvenile morphologies
are important in order to obtain a better understanding of
evolutionary ecology and phylogeny (Yamaguchi et al.
2000). Accordingly, the present study aimed to describe the
morphological development of the early life stages of this
species in detail using a series of laboratory-reared larval
and juvenile H. malcolmi.
Materials and methods
Parental fishes and egg collection. Broodstocks of Hypsi-
barbus malcolmi were obtained from the National Aqua-
culture Development Center, Department of Livestock and
Fisheries, Namxuang, Laos, on 25 May 2009. An LH-RH-
analog hormone (Suprefact: Hoechst AG Inc., Germany) in
combination with a dopamine inhibitor (Motilium: Olic
Ltd., Thailand) was injected once at the rate of 1 ml/1 kg
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Y. Ogata et al.
FW (fish wet weight) for the former and 500 mg/1 kg FW
for the latter into three males (250 g) and one female
(1,172 g) at 1700 hours on the same day. The water tem-
perature during the induced breeding period was
27.0–28.0°C. Some 100,000 fertilized eggs were spawned
at 2200 hours on the same day. Eggs were subsequently
incubated in a round hatching jar (30 cm diameter, 40 cm
height) with aeration. Water temperature during egg incu-
bation was 27.9–29.0°C.
Rearing of larvae and juveniles. Hatching took place on
26 May 2009 at 0600 hours, with some 85,000 larvae
being obtained. Of these, nine hundred were stocked in
three circular aquaria (300 larvae in each aquarium,
labelled I, II and III), each containing 30 l of water (35 cm
diameter 9 35 cm depth, each), and reared under ambient
water temperatures ranging from 25.4 to 30.0°C (mean
27.9°C). Starting on day 2 after hatching and continuing
until day 12, the fish were fed Brachionus angularis
(5 individuals/ml). Artemia sp. nauplii (0.5 individuals/ml)
were added from day 6 to day 13, and wild copepoda and
Moina sp. (0.1 individuals/ml) from day 13 to day 30. The
densities of the plankton provided were monitored and
maintained at a stable level three times a day. Fish samples
for morphological observations were collected from aqua-
ria I and II, and the survival rate (%) was estimated as the
number of fish remaining relative to the number initially
stocked (=300) from aquarium III.
Observations and measurements. Fertilized eggs were
collected 8 h after spawning (just before hatching), and
larvae and juveniles on days 0–6, 8, 10, 12, 14, 16, 19, 22,
25, 28 and 30 after hatching [larvae on days 0 (day 0 cor-
responded to newly hatched larvae) and 1 were collected
twice a day, i.e., at 1 h (day 0-i) and 9 h (day 0-ii), 27 h (day
1-i) and 33 h (day 1-ii) after hatching, respectively]. These
were preserved in 5% formalin immediately after sampling.
Observations and measurements were made at the Fisheries
Division, Japan International Research Center for Agricul-
tural Sciences, Tsukuba, Japan, and the fertilized eggs and
some fish samples were registered in the Museum of Tokyo
University of Marine Science and Technology (MTUF).
Measurements were performed on seven eggs [MTUF-
P(L)-24522] and 8–10 fish collected on each day of sam-
pling, totaling 153 larvae (1.9–11.5 mm in body length) and
26 juveniles (10.4–12.9 mm in body length). These were
used for observations of general morphology, number of
myomeres, pigmentation and fin development, and the
following morphometric measurements (in mm): body
length (BL), head length (HL) and maximum body depth
(BD) from day 0-i, snout length (SnL), eye diameter (ED)
and pre-anal length (PAL) from day 0-ii (after appearance),
and upper jaw length (UJL) from day 1-ii (after mouth
opening). Feeding incidence rates [=(number of larvae with
zooplankton in gut)/(number of observed larvae) 9 100%]
Morphological development of Hypsibarbus malcolmi
were observed in day 0–3 larvae in order to ascertain the
timing of the onset of feeding. Body length was taken as the
notochord length before hypural formation, and the stan-
dard length thereafter. Since the yolk shape was initially
elliptic, becoming conic and/or columnar with decreasing
volume, yolk volumes (mm3, V) were estimated from
the formulae V = 4/3 9 L 9 H 9 W 9 p when elliptic,
V = 1/3 9 (H/2)2 9 L when conic, and V = (H/2)2 9
L when columnar, where L, H and W denote yolk length
(mm), yolk height (mm) and yolk width (mm). Some
specimens were stained in alizarin red S and/or Alcian
blue 8GX for observations of fin ray formation and jaw
development. Fourteen specimens were sketched [MTUF-
P(L)-22112–22122 and MTUF-P(L)-24519–24521]. Egg
diameters were measured 8 h after spawning. Develop-
mental stages recognized in the present study were deter-
mined as yolksac, preflexion, flexion, postflexion and
juvenile, following Kendall et al. (1984), since the yolk was
still present at the early flexion stage in this species. Mea-
surement methods followed Leis and Trnski (1989).
Results
Spawning and hatching. Spawning took place at
2200 hours on 25th May 2009, with ca. 100,000 fertilized
eggs being obtained (fertilization rate ca. 70%). Eggs were
pelagic and almost spherical in shape, the egg diameter
ranging from 3.1 to 3.4 (mean ± SD: 3.3 ± 0.1) mm
(n = 7). Hatching took place at 0600 hours on 26 May
2009, 8 h after spawning.
Larvae and juveniles. General morphology. The BL of
day 0-i larvae (1 h after hatching) ranged from 1.9 to 2.2
(mean ± SD: 2.0 ± 0.2) mm (n = 8), reaching 6.1 ±
0.4 mm (n = 10) on day 10, 10.1 ± 0.5 mm (n = 10) on
day 19 and 12.1 ± 0.9 mm (n = 10) on day 30 (Fig. 1). Of
the 300 fish initially stocked in aquarium III, 278 were
Fig. 1 Relationship between hours/days after hatching and body
length (mm) in laboratory-reared larval and juvenile Hypsibarbus
malcolmi from day 0 to day 30 after hatching. Solid circles means,
vertical bars standard deviations
391
Table 1 Body length (BL) and age in days afor each developmental
stage of Hypsibarbus malcolmi
Stage BL (mm) Age (days) n
Yolksac larvae 1.9–3.3 0–2 39
Preflexion larvae 3.2–5.3 3–10 44
Flexion larvae 5.2–8.2 8–14 34
Postflexion larvae 8.5–11.5 14–30 36
Juveniles [10.4 [19 26
collected on day 30 after hatching. Thus, the survival rate
during the 30 days following hatching was estimated as
92.7%.
The BLs of larvae and juveniles at each developmental
stage are shown in Table 1. Yolksac larvae on day 0-i had a
large oval yolksac [vertical axis 1.51 ± 0.07 (mean ± SD)
mm, horizontal axis 0.23 ± 0.04 mm (n = 8)], extending
ca. 75% of the BL, with its anterior margin bordering the
ventral aspect of the bent head (Fig. 2a); yolk volume
(V, mm3) rapidly decreased linearly during days 0–2
(Figs. 2a–e, 3), being completely absorbed in preflexion
larvae by day 3 (3.5 ± 0.1 mm BL, n = 10; Figs. 2f, 3).
The feeding incidence rate (%) was 0 until day 1-ii, with
feeding being ascertained in two of nine specimens in
which the yolk had been completely absorbed on day 2
(feeding incidence rate 22%), although the yolk remained
in the other seven specimens (Fig. 2e); following yolk
absorption in all specimens (on day 3, Fig. 2f), the feeding
incidence rate reached 100%.
The head was initially bent, and it subsequently sepa-
rated from the anterior margin of the yolksac in yolksac
larvae on day 1-i (3.1 ± 0.1 mm BL, n = 8; Fig. 2c). All
myomeres were observable in yolksac larvae on day 0-ii
(2.8 ± 0.1 mm BL, n = 8), with preanal and postanal
myomere counts on that day being 23 ? 9 = 32 (Fig. 2b),
although they were subsequently 20–21 ? 11–12 = 31–33
(Fig. 2c–m); myomeres were invisible in juveniles
(Fig. 2n). The ventral finfold initially originated on the
anterior 41–43% BL (Fig. 2a), and divided into anterior
and posterior portions (relative to the anus) in yolksac
larvae on day 1-i (Fig. 2c), with the latter forming the anal
fin anlage in day 8 flexion larvae [5.3 mm BL (Fig. 2h),
and both portions disappearing in juveniles (days 22–30;
Fig. 2n). The dorsal finfold initially originated on the
anterior 18–20% BL (Fig. 2a), forming the dorsal fin
anlage in day 8 flexion larvae [5.3 mm BL (Fig. 2h), and
disappearing in day 16 postflexion larvae [8.9 mm BL
(Fig. 2j); the caudal finfold was becoming fan-shaped in
day 0-ii yolksac larvae (Fig. 2b), becoming forked in day
10 flexion larvae [6.0 mm (Fig. 2i), and disappearing in
juveniles (days 22–30; Fig. 2n).
The mouth and anus opened in yolksac larvae on day
1-i, with the posterior limit of the mouth initially being
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Fig. 2 Laboratory-reared larval and juvenile Hypsibarbus malcolmi
from days 0-i–30 after hatching. a Yolksac larva, day 0-i [1.9 mm BL,
MTUF-P(L)-22112]; b yolksac larva, day 0-ii [2.9 mm BL, MTUF-
P(L)-22113]; c yolksac larva, day 1-i [3.1 mm BL, MTUF-P(L)-
22114]; d yolksac larva, day 1-ii [3.0 mm BL, MTUF-P(L)-22115];
e yolksac larva, day 2 [3.3 mm BL, MTUF-P(L)-22116]; f preflexion
larva, day 4 [3.5 mm BL, MTUF-P(L)-22117]; g preflexion larva, day
located beneath the center of the eye (Fig. 2c), and then
beneath the anterior eye margin throughout all subsequent
developmental stages (Fig. 2d–n); the upper and lower
jaws formed in yolksac larvae on day 2 (3.3 ± 0.1 mm BL,
n = 9; Fig. 2e). A nostril formed between the upper jaw tip
and the eye in day 4 preflexion larvae (3.7 ± 0.2 mm BL,
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Y. Ogata et al.
6 [4.4 mm BL, MTUF-P(L)-22118]; h flexion larva, day 8 [5.6 mm
BL, MTUF-P(L)-22119]; i flexion larva, day 10 [6.3 mm, MTUF-
P(L)-22120]; j flexion larva, day 12 [7.3 mm BL, MTUF-P(L)-22121];
k postflexion larva, day 14 [7.6 mm BL, MTUF-P(L)-221122];
l postflexion larva, day 16 [9.1 mm BL, MTUF-P(L)-24519]; m post-
flexion larva, day 22 [10.1 mm BL, MTUF-P(L)-24520]; n juvenile,
day 30 [12.9 mm BL, MTUF-P(L)-24521]
n = 10; Fig. 2f), with its shape changing from oval to
slender (gourd-shaped) with growth (Fig. 3f–m), and
dividing into two in juveniles (days 22–30; Fig. 2n).
The operculum appeared in yolksac larvae on day 1-i
(Fig. 2c), the lateral line in flexion larvae on day 4
(3.7 ± 0.2 mm BL, n = 10; Fig. 2f), and the gas bladder
Morphological development of Hypsibarbus malcolmi
Fig. 3 Relationship between hours/days after hatching and yolk
volume (mm3) in laboratory-reared larval Hypsibarbus malcolmi from
day 0 to day 3 after hatching. Solid circles means, vertical bars
standard deviations. V and T are yolk volume and hours after
hatching, respectively
between the abdominal cavity and notochord (extending
from the 3–4th to 8–9th myomeres) in flexion larvae on day
2 (Fig. 2e). The notochord subsequently became invisible
in day 10 flexion [6.0 mm BL (Fig. 2i).
HL initially measured 14–16% BL, with the proportion
subsequently decreasing slightly to 13–14% BL in yolksac
larvae \3.0 mm BL (day 0-ii), but increasing to 30–36%
BL in day 22 juveniles [10.4 mm BL and becoming
constant thereafter (Fig. 4a). ED varied from 3–6% BL in
yolksac larvae on day 0-i (at eye formation) to preflexion
larvae on day 5 (3.8 ± 0.2 mm BL, n = 9), with the pro-
portion subsequently increasing to 9–11% BL in day 22
juveniles [10.4 mm BL (Fig. 4b) and becoming constant
thereafter. SnL was ca. 2% BL in yolksac larvae on day 0-i,
increasing to 6–7% BL in day 19 postflexion larvae
[9.3 mm BL, and remaining constant thereafter (Fig. 4c).
BD was initially 17–20% BL, with the proportion rapidly
decreasing to 10–12% BL with yolk absorption and body
extension in day 2 yolksac larvae \3.3 mm BL and sub-
sequently increasing to 27–30% BL in day 22 juveniles
[10.4 mm BL (Fig. 4d). PAL varied from 60 to 70% BL
in yolksac larvae on day 1-i, becoming constant (ca. 70%)
thereafter (Fig. 4e). UJL was 4–5% BL at mouth opening
in yolksac larvae on day 1-ii, with the proportion subse-
quently increasing to 10–12% BL in day 14 postflexion
larvae [8.4 mm BL and becoming constant thereafter
(Fig. 4f).
Fin development. Dorsal fin soft rays appeared in day 10
flexion larvae [6.0 mm BL (Figs. 2i, 5a) and spiny soft
rays in day 14 postflexion larvae [8.0 mm BL, with the
full complement (iv, 8) being attained in day 22 postflexion
larvae [10.2 mm BL (Figs. 2l, 5a). The first spiny soft ray
was short and subcutaneous, but observable by alizarin red
staining; soft ray segmentation initiated in day 12 flexion
larvae [7.3 mm BL (Fig. 2j) and was completed (eight
393
segmented rays) in day 22 postflexion larvae [10.1 mm
BL (Fig. 2m); soft ray branching was initiated in day 16
postflexion [9.6 mm BL, and was completed (seven
branched rays) in day 30 juveniles [12.2 mm BL
(Fig. 2n). Anal fin soft rays appeared in day 10–12 flexion
larvae [7.1 mm BL (Figs. 2j, 5b), with the full comple-
ment (iii, six) being attained in day 14 flexion lar-
vae [7.6 mm BL (Figs. 2k, 5b); soft ray segmentation was
initiated in day 16 postflexion larvae [9.6 mm BL and
completed (six segmented rays) in day 19 postflexion lar-
vae [9.9 mm BL (Fig. 2m); soft ray branching initiated in
day 16 juveniles [9.0 mm BL (Fig. 2l) and completed (six
branched rays) in day 30 juveniles [12.2 mm BL
(Fig. 2n). Pectoral fin soft rays appeared in day 12–14
flexion larvae [7.4 mm BL (Figs. 2k, 5c), with the full
complement (14–15) being attained in juveniles (Figs. 2n,
5c); soft ray segmentation was initiated in day 16 post-
flexion larvae [9.1 mm BL (Fig. 2l) and completed (9–12
segmented rays) in juveniles [11.5 mm BL (Fig. 2n);
branched soft rays were absent. Pelvic fin soft rays
appeared in day 12–14 flexion larvae [7.6 mm BL
(Figs. 2k, 5d) and attained the full complement (i, eight) in
day 16 postflexion larvae [8.7 mm BL (Fig. 2l); soft ray
segmentation was initiated in days 14–16 postflexion lar-
vae [8.4 mm BL (Fig. 2l) and completed (6–7 segmented
rays) in juveniles; soft ray branching was initiated in day
22 postflexion larvae [10.1 mm BL (Fig. 2m) and com-
pleted (six branched rays) in juveniles (Fig. 2n). Caudal fin
soft rays appeared in day 8 flexion larvae [4.6 mm BL
(Figs. 2h, 5e), with the principal soft rays attaining the
full complement (10 ? 10 = 20) in day 12 flexion lar-
vae [6.5 mm BL (Fig. 2j), and total soft rays (including
procurrent rays) attaining the full complement
(18–20 ? 16–17 = 35–38) in days 19–22 postflexion lar-
vae [10.1 mm BL (Figs. 2m, 5e); soft ray segmentation
was initiated in day 16 postflexion larvae [6.1 mm BL
(Fig. 2i) and completed (19 segmented rays) in day 22
postflexion larvae [10.1 mm BL (Fig. 5m); soft ray
branching was initiated in day 22 postflexion lar-
vae [10.1 mm BL (Fig. 2m) and completed (16–17 bran-
ched rays) in juveniles (Fig. 2n).
Pigmentation. Melanophores were entirely absent in day
0-i larvae (Fig. 2a). Melanophore deposition on the eye
started in day 1-ii yolksac larvae (Fig. 2d), with the eye being
fully pigmented in yolksac larvae on day 2 (Fig. 2d–e). One
to two stellate melanophores were present mid-dorsally on
the head in day 6 preflexion larvae (4.4 mm BL; Fig. 2g),
thereafter increasing in number and becoming punctuate,
and covering the entire dorsal surface of the head by the
juvenile stage (Fig. 2n). Two to three light stellate melano-
phores appeared on the operculum in day 6 preflexion larvae
(Fig. 2g), and slightly increased in number by the juvenile
stage (Fig. 2n).
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Fig. 4 Proportions of a head length (HL), b eye diameter (ED), c snout length (SnL), d body depth (BD), e preanal length (PAL) and f upper jaw
length (UJL) to body length (BL) in laboratory-reared larval and juvenile Hypsibarbus malcolmi
Many small light stellate melanophores were initially
present on the dorsal surface of the gas bladder and on the
bordering aspect between the intestine and abdominal trunk
in day 4 preflexion larvae (3.5 mm BL; Fig. 2f), with the
former disappearing in day 10 flexion larvae (6.3 mm BL;
Fig. 2i) and the latter increasing in size but decreasing in
number and becoming invisible with growth by day 16
postflexion larvae (9.1 mm BL; Fig. 2l).
A series of small punctate melanophores appeared on
the dorsal surface of the trunk in day 12 flexion larvae
(7.3 mm BL; Fig. 2j), and subsequently increased in
number and extended more posteriorly to the dorsal sur-
face of the caudal body (Fig. 2k–m) with growth. Stellate
melanophores apparent posteriorly on the lateral line in
day 12 flexion larvae (7.3 mm BL; Fig. 2j), gradually
increased in number and extended anteriorly with growth
(Fig. 2k–m). Few lateral melanophores were apparent on
the bodies of postflexion larvae on day 22 (Fig. 2m),
although several punctate and stellate melanophores were
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Y. Ogata et al.
evident thereafter; they increased rapidly and covered the
entire upper body above the lateral line by the juvenile
stage (Fig. 2n). Several stellate melanophores apparent on
the abdominal region in day 8 flexion larvae (5.6 mm BL;
Fig. 2h) increased slightly in number and remained until
the juvenile stage (Fig. 2n). Two or three stellate mela-
nophores on the posteriormost portion of the body in day
10 flexion larvae (6.3 mm BL) gradually increased sub-
sequently (Fig. 2j–n).
Small light punctate and stellate melanophores appar-
ent on the caudal fin in day 10 flexion larvae (6.3 mm
BL; Fig. 2i) increased in number and covered the entire
caudal fin by the juvenile stage (Fig. 2n). Punctate and
stellate melanophores were also apparent anteriorly on the
dorsal and anal fins in day 14 postflexion larvae (7.6 mm
BL; Fig. 2k), the former increasing in number with
growth, and the latter disappearing by the juvenile stage
(Fig. 2n). Melanophores were absent on pectoral and
pelvic fins.
Morphological development of Hypsibarbus malcolmi
Fig. 5 Relationships between body length (mm) and fin ray numbers
in laboratory-reared larval and juvenile Hypsibarbus malcolmi.
a Dorsal fin (DFR), b anal fin (AFR), c pectoral fin (P1FR),
d pelvic fin (P2FR), e caudal fin (CFR, closed circles principal fin ray
number, opened circles total fin ray number)
395
Discussion
In Hypsibarbus malcolmi, no barbels were observed in the
specimen of 12.9 mm BL, which was the largest fish used
in the present study. However, only a single pair is present
in larger juveniles (ca. 35.0–40.0 mm BL), and two pairs
on the upper jaw in adults (Rainboth 1996a). These suggest
that a pair develops at between 13.0 and 35.0 mm SL, and
another pair thereafter. In other cyprinids, it was reported
that barbels develop at 23–25 mm SL in Rhinichthys cat-
aractae (see Cooper 1980) and ca. 17 mm SL in Candidia
barbatus (see Sado and Kimura 2002b).
The body length of newly hatched H. malcolmi larvae
observed here was ca. 2 mm (Fig. 1). This size would place
it into the group of cyprinids with smaller newly hatched
larvae [e.g., 2.3–2.6 mm BL in Horadandia atukokari (see
Sado and Kimura 2005a), 2.2–2.6 mm BL in Tanichthys
albonubes (see Sado and Kimura 2005c)], in contrast to the
group with larger newly hatched larvae [e.g.,[4 mm SL in
Ctenopharyngodon idella, Hypophthalmichthys molitrix
and Hypophthalmichthys nobilis (see Yi et al. 2006), ca.
5 mm BL in Zacco temminckii (see Sado and Kimura
2002a) and Barilius canarensis (see Sado and Kimura
2005b)]. In the present study, a high survival rate ([90%)
was realized during the initial 30-day post-hatching period
using the locally occurring rotifer Brachionus angularis in
Laos as the initial dietary plankton. Since the feeding of
fish larvae is dependent on the size relationship between
gape and prey (Bremigan and Stein 1994), the H. malcolmi
larvae at the onset of feeding (mean ± SD: 3.3 ± 0.1 mm
BL) may not be able to feed on larger zooplankton, as
reported for Anabas testudineus, which is small when
newly hatched (\2.0 mm BL) and not capable of feeding
on large zooplankton at the onset of feeding (ca. 3.0 mm
BL) (Morioka et al. 2009b). Therefore, in order to produce
H. malcolmi seed in mass quantities, the stable cultivation
of small-sized zooplankton (such as rotifers) is considered
to be required. However, artificial rotifer production is not
currently established in Laos, except in small-scale
experimental production (Morioka et al. 2009b), and only
large-sized zooplankton (e.g., Moina spp.) is cultured on a
commercial basis and is stably supplied in the region.
The onset of feeding occurred in day 2–3 larvae
immediately after complete yolk absorption and gas blad-
der formation (Fig. 2e–f), following the jaw formation and
eye pigmentation observed in day1-ii yolksac larvae
(Fig. 2d). This demonstrated that such developments in
morphology are necessary processes for the onset of
feeding, and the preparatory period for the shift from
endogenous to exogenous energy-dependent periods was
short: less than 1 day under the experimental protocol of
123
396
this study. Furthermore, this short preparatory period
illustrates that initial feeding success is an important factor
that influences early larval survival in H. malcolmi. The
shift from endogenous to exogenous energy-dependent
periods has been examined in a number of marine fish
species, with aspects of development and survival during
the larval stages being investigated (Kohno 1998; Moteki
et al. 2001), but similar investigations of freshwater species
are still limited despite several reports on morphological
development in early life stages, such as for Rhinichthy
cataractae and Nocomis micropogo (see Cooper 1980),
Barilius canarensis (see Sado and Kimura 2005a),
Tanichthys albonubes (see Sado and Kimura 2005c), and
Inlecypris auropurpureus (see Sado and Kimura 2006).
Apart from these, however, similar achievements to the
present study have been presented in recent years during
searches for improved seed productivity of, for example,
North African catfish Clarias gariepinus (Clariidae, see
Matsumoto et al. 2001), climbing perch Anabas testudineus
(Anabantidae, see Morioka et al. 2009a), snakeskin gou-
rami Trichogaster pectoralis (Osphronemidae, see Morioka
et al. 2010a), and sutchi catfish Pangasianodon hypoph-
thalmus (Pangasiidae, see Morioka et al. 2010b). The
preparatory periods during the energy-source changeover
for the aforementioned five species were ca. 5, 5, 10, [1
and [2 days, respectively, with those for the former three
species being much longer than that for H. malcolmi. This
suggests that the changeover from endogenous to exoge-
nous energy-dependence progressed more quickly and
smoothly in H. malcolmi and P. hypophthalmus than in the
former three species, but earlier feeding success is more
important in H. malcolmi and P. hypophthalmus for their
subsequent survival. The duration of yolk absorption with
respect to the onset of feeding is considered to be an
essential feature of any examination of survival strategies
during early life stages that are relevant to the evolutionary
ecology and phylogenetic relationships in Cyprinidae,
since the genus Hypsibarbus and related genera (e.g.,
Barbodes and Propuntius) (see Rainboth 1996a) include a
number of species with global distributions, although they
are centered on Southeast Asia.
Despite the rich indigenous fish diversity in Indochinese
freshwaters, particularly of Cyprinidae, the early morphol-
ogy of the latter has only rarely been described so far, due to
a lack of laboratory-based seed production techniques,
although this has been achieved for several exotic cypri-
nid species that are now established in the region (e.g.,
Ctenopharyngodon idella, Hypophthalmichthys molitrix,
H. nobilis; see Yi et al. 2006). However, the number of
indigenous cyprinid species to which artificial seed pro-
duction can be applied has increased with recent techno-
logical progress (e.g., Barbonymus gonionotus, Cirrhinus
microlepis, Cirrhinus molitorella, Probarbus jullieni, Labeo
123
Y. Ogata et al.
chrysophekadion), and further progress on morphological
descriptions of larval and juvenile stages should be made. In
addition, considering the species confusion among mor-
phologically similar cyprinids (e.g., the genera Hypsibarbus
and Barbonymus) in aquaculture farms in the region
(Vidthayanon 2001), species identification of broodstocks
and descriptions of larval and juvenile morphologies for
each species are urgently required for further aquacultural
development. Furthermore, because of potential future
declines in regional fish diversity, including that of cypri-
nids, owing to rapid population growth of invasive alien
species and their settlement in the region and increased
fishing efforts, morphological descriptions of such indige-
nous species are indispensable for stock assessments as well
as phylogenetic and evolutionary ecological studies.
Acknowledgments We express our sincere gratitude to all of the
staff of the project of the JICA (Japan International Cooperation
Agency) at the National Aquaculture Development Center, Ministry
of Agriculture and Forestry, Laos. Species identification by
K. Shibukawa and K. Utsugi, Nagao Natural Environment Founda-
tion, Japan, was deeply appreciated. Our thanks also to G. Hardy for
his constructive English revision of our paper.
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