Ichthyol Res (2012) 59:5362
DOI 10.1007/s10228-011-0256-9
FULL PAPER
Growth and morphological development of laboratory-reared
larval and juvenile three-spot gourami Trichogaster trichopterus
Shinsuke Morioka Phonaphet Chanthasone
Phoutsamone Phommachan Bounsong Vongvichith
Received: 17 May 2011 / Revised: 14 October 2011 / Accepted: 15 October 2011 / Published online: 25 November 2011
The Ichthyological Society of Japan 2011
Abstract Morphological development, including the body
proportions, fins, pigmentation and labyrinth organ, in lab-
oratory-hatched larval and juvenile three-spot gourami
Trichogaster trichopterus was described. In addition, some
wild larval and juvenile specimens were observed for com-
parison. Body lengths of larvae and juveniles were
2.5 0.1 mm just after hatching (day 0) and 9.2 1.4 mm
on day 22, reaching 20.4 5.0 mm on day 40. Aggregate fin
ray numbers attained their full complements in juveniles
[11.9 mm BL. Preflexion larvae started feeding on day 3
following upper and lower jaw formation, the yolk being
completely absorbed by day 11. Subsequently, oblong con-
ical teeth appeared in postflexion larvae [6.4 mm BL (day
13). Melanophores on the body increased with growth, and a
large spot started forming at the caudal margin of the body in
flexion postlarvae[6.7 mm BL, followed by a second large
spot positioned posteriorly on the midline in postflexion
larvae [8.6 mm BL. The labyrinth organ differentiated in
postflexion larvae [7.9 mm BL (day 19). For eye diameter
and the first soft fin ray of pelvic fin length, the proportions in
laboratory-reared specimens were smaller than those in wild
specimens in 18.524.5 mm BL. The pigmentation pattern
of laboratory-reared fish did not distinctively differ from that
in the wild ones. Comparisons with larval and juvenile
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
P. Chanthasone  P. Phommachan  B. Vongvichith
Aquaculture Research Unit, Living Aquatic Resource Research
Center, National Agriculture and Forestry Research Institute,
Khounta Village, Sikhotabong District, Vientiane, Lao PDR
morphology of a congener T. pectoralis revealed several
distinct differences, particularly in the numbers of myo-
meres, pigmentations and the proportional length of the first
soft fin ray of the pelvic fin.
Keywords Trichogaster trichopterus  Larvae 
Juveniles  Morphology
Introduction
The three-spot gourami Trichogaster trichopterus (Family
Osphronemidae, Suborder Anabantoidei) is one of the most
common air-breathing freshwater fishes, occurring naturally
in the Mekong Basin of Laos, Yunnan, Thailand, Cambodia
and Vietnam (Rainboth 1996; Kottelat 1998, 2001), and
elsewhere because of accidental or deliberate introductions,
including the Philippines, Papua New Guinea, Sri Lanka,
Columbia and the Dominican Republic, where breeding
populations of the species have now been established (Lever
1996; Welcomme 1988). The species is often found in rice
paddies, or ditches and streams with dense vegetation
(Rainboth 1996), and is a valuable food source for local
inland communities as well as a trade target in the orna-
mental fish market (Rainboth 1996; Iwata et al. 2003).
In Laos, ten anabantoid species are known to occur,
including nine species of Osphronemidae, contained in the
genera Betta, Macropodus, Osphronemus, Trichogaster
and Trichopsis, and one of Anabantidae, in the genus
Anabas (see Kottelat 2001). These species are all signifi-
cant targets of commercial and small-scale fisheries in the
region (Iwata et al. 2003; Welcomme and Vidthayanon
2003), although differences in marketable values exist
according to species and size. On the other hand, the
human population has grown rapidly in the region, and
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environmental changes, such as urbanization, increasing
land use for crops and biological invasions by aliens (e.g.,
Oreochromis spp., Hypophthalmichthys molitrix, Aristich-
thys nobilis, etc.) and hybrid fishes (cross-bred Clarias
spp.) (Iwata et al. 2003; Welcomme and Vidthayanon
2003; Na-Nakorn et al. 2004; Senanan et al. 2004), have
occurred. Such impacts, disturbing regional fish diversity,
have the potential to lead to declines of indigenous fish
stocks, including Anabantoidei. Accordingly, stock
assessments of these species are required immediately,
with unequivocal species identification procedures, partic-
ularly of larval and juvenile stages, being very important.
Hence, accumulation of early life history information is
essential, including morphological descriptions of indige-
nous species. Furthermore, since Anabantoidei has long
been a systematically contentious taxonomic group, owing
to species diversification and their evolutionary ecology
(Ruber et al. 2006), descriptions of early life stage mor-
phology should provide significant new information, only
Anabas testudineus and Trichogaster pectoralis having
been thus treated so far (Morioka et al. 2009, 2010a). The
objective of this study, therefore, was to facilitate the
identification of early life stages of the three-spot gourami
Trichogaster trichopterus, the morphology of larvae and
juveniles being described in detail from a series of labo-
ratory-reared specimens. In addition, some wild larval and
juvenile specimens were observed for morphological
comparisons with laboratory-reared fish.
Materials and methods
Parental fishes and egg collection. Broodstocks of Tric-
hogaster trichopterus were collected from small water
bodies in the Nam Xuang area, 40 km north of Vientiane
City, Laos, in February 2009. Broodstocks were reared in
the Living Aquatic Resources Research Center, Vientiane
City, Laos. A LH-RH-analogue hormone (Suprefact:
HOECHST AG Inc., Germany) was injected twice in
combination with a dopamine inhibitor (Motilium: OLIC,
Ltd., Thailand) at the rate of 2 mg/100 g FW (fish wet
weight) for the former and 1 mg/100 g FW for the latter,
into 5 males (body weight ca. 60 g) and 4 females (ca.
50 g) at 1200 and 1630 hours on 14 May 2010, prior to
placing them in a spawning tank containing 100 l water.
Because Trichogaster species are known to form a bubble
nest beneath floating aquatic plants, eggs being spawned in
the former (Cole et al. 1999), some taro leaves (Family:
Araceae; ca. 2530 cm diameter) were floated in the
spawning tank. Water temperature during the induced
breeding period ranged from 32.8 to 34.0C.
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S. Morioka et al.
Rearing larvae and juveniles. Fertilized eggs were
obtained through the induced breeding process, the newly
hatched larvae being separated into two rectangular aquaria
(30 cm length 9 40 cm width 9 30 cm depth containing
36 l of water each) and reared under ambient water tem-
peratures ranging from 25.8 to 32.8C (mean SD
28.6 1.2C). From day 2 after hatching, fish were fed
wild zooplankton (e.g., Cyclops, Moina, Brachionus) col-
lected from a fertilized earthen pond at a density of 1015
individuals/ml in the rearing tanks three times a day until
day 11. From days 940 inclusively, Artemia nauplii were
also added at a density of 23 individuals/ml three times a
day.
Observations and measurements. Fertilized eggs were
collected ca. 1 h after spawning, and larvae and juveniles
on days 05, 7, 9, 11, 13, 16, 19, 22, 25, 30, 35 and 40 after
hatching. All were preserved in 5% formalin immediately
after sampling. Observations and measurements were made
in the Fisheries Division of Japan International Research
Center for Agricultural Sciences, Tsukuba, Japan, the fer-
tilized eggs and some fish samples being registered at the
Tokyo University of Marine Science and Technology
(MTUF) Museum. Measurements were made on 10 eggs
[MTUF-P(L)-26590] and 5 fish on each day of sampling,
totaling 76 larvae [2.411.9 mm in body length (BL)] and
19 juveniles (11.926.1 mm in BL). Of these, 7 were dis-
sected for observations of the labyrinth organ [MTUF-
P(L)-2660326609], the remainder (n = 88) being used for
observations on general morphology, number of myo-
meres, pigmentation, fin development and the following
morphometric measurements (mm): BL, head length (HL),
pre-anal length (PAL), maximum body depth (BD), eye
diameter (ED), snout length (SnL), upper jaw length (UJL)
and length of the first soft ray of pelvic fin (P2FSR). HL,
SnL, PAL and UJL were measured from day 1 after
operculum-jaw formation and anus opening, P2FSR from
its appearance on day 22 and the remainder from day 0.
Further, proportions and pigmentations of nine wild fish
(one postflexion larva and eight juveniles; 11.624.5 mm
BL) collected from the Nam Xuang area on 22 July 2011
were observed for comparisons between laboratory-reared
and wild fishes. BL was taken as notochord length before
hypural formation, and standard length thereafter. Some
specimens were stained in alizarin red S or alcian blue 8GX
for observations of fin ray formation and jaw development.
Thirteen laboratory-reared and three wild specimens were
sketched [MTUF-P(L)-24715, 24716, 2659126600 and
26610 for the former and MTUF-P(L)-24717, 24718,
24720 for the latter]. Egg diameter was measured after
spawning. Measurement methods followed Leis and Trnski
(1989).
Morphological development of Trichogaster trichopterus 55

Results cavity beside upper base of pectoral fin (Fig. 2cg), com-
pletely absorbed in flexion larvae by day 11 (5.3 0.6 mm
Spawning and hatching. Spawning took place approxi- BL, n = 5) (Fig. 2h). Onset of feeding was observed in day
mately 1314 h after hormone injection, at 05300630 hours 3 preflexion larvae (3.8 0.2 mm BL, n = 5).
on 15 May 2010, the spawned eggs being concentrated in the Head initially bent with ventral aspect bordering on ante-
bubble nest beneath taro leaves, and approximately 15,000 rior margin of yolk sac (Fig. 2a), subsequently stretching and
fertilized eggs were obtained. Eggs were isolated, epipelagic separating from the yolk sac from day 1 (3.1 0.1 mm BL,
and slightly oval in shape, their diameter ranging from 0.90 to n = 5) (Fig. 2b); operculum appearing on day 1 (Fig. 2b);
0.97 (0.92 0.02) mm (n = 10) in maximum axis and 0.77 lateral line appearing on day 2 (Fig. 2c). Myomeres fully
to 0.93 (0.85 0.04) mm (n = 10) in minimum axis. observable (numbering 78 ? 2526 = 3234) in yolk sac
Hatching took place approximately 24 h after spawning at larvae[4.1 mm BL (day 7; Fig. 2f), subsequently becoming
0600 hours on 16 May 2010. invisible in postflexion larvae[8.5 mm BL (day 19; Fig. 2j).
Laboratory-reared larvae and juveniles. General Gas bladder visible between abdominal cavity and notochord
morphology. The BL (mm) of newly hatched larvae (day 0) in preflexion larvae on day 9 (4.5 0.5 mm BL, n = 10;
ranged from 2.4 to 2.5 (2.5 0.1) mm (n = 5), reaching Fig. 2g), becoming invisible in postflexion larvae [8.2 mm
4.2 0.3 mm on day 5 (n = 5), 5.3 0.6 mm on day 11 BL on day 16 (Fig. 2j).
(n = 5), 9.2 1.4 mm on day 22 (n = 5), 16.5 4.7 mm Ventral fin fold originating on anterior 4648% BL
on day 35 (n = 5) and 20.4 5.0 mm on day 40 (n = 5) (Fig. 2a), divided into anterior and posterior portions rel-
(Fig. 1). BLs of larvae and juveniles at each developmental ative to anus position in day 1 larvae (Fig. 2b), anterior
stage are shown in Table 1. portion disappearing in postflexion larvae [10.6 mm BL
Newly hatched larvae (n = 5) with a large oval yolk sac (day 25; Fig. 3a), posterior portion in juveniles (Fig. 3b);
(vertical axis 0.99 0.03 mm, horizontal axis dorsal fin fold originating on anterior 3234% of body
0.72 0.05 mm); anterior margin of yolk sac bordering on (Fig. 2a), disappearing in postflexion larvae [10.0 mm BL
ventral aspect of bent head (Fig. 2a); yolk split into two (day 25; Fig. 3a); caudal fin fold initially round, fan-shaped
portions in day 2 preflexion larvae (3.4 0.1 mm BL, (Fig. 2a), disappearing in juveniles (Fig. 3b). Pterygio-
n = 5) (Fig. 2c); yolk moving dorsally to above abdominal phores of anal fin clearly visible in flexion larvae [6.5 mm
BL (Fig. 2i) and becoming invisible in juveniles (Fig. 3b).
Mouth and anus opened in day 1 larvae (Fig. 2b), mouth
reaching a vertical through anterior ca. 40% of ED on day
1, subsequently posterior edge of mouth moving forward
with growth, located in anterior half of snout in juvenile
stage (Figs. 2cj, 3a, b); upper and lower jaws formed in
day 1 larvae (Fig. 2b); a few sharp, oblong conical teeth
appearing on both jaws in postflexion larvae [6.4 mm BL
(day 13), teeth increasing in number with growth. Nostril
appearing in day 3 larvae (Fig. 2d), dividing into two in
juveniles [13.5 mm BL (Fig. 3b, c).
Proportions. Head length 1619% BL in day 1 larvae
(Fig. 4a), proportion subsequently increasing with growth,
reaching ca. 3336% BL in postflexion larvae [9.4 mm
Fig. 1 Changes in body length (mm) of laboratory-reared larval and BL (day 22; Fig. 4a). PAL 3841% BL initially in day 1
juvenile three-spot gourami Trichogaster trichopterus from days 0 to
40 after hatching. Vertical bars indicate standard deviations larvae (Fig. 4b), proportion decreasing to 3537% BL with
tail extension in flexion larvae \5.2 mm BL (day 9), sub-
sequently increasing and reaching to 4952% BL in post-
flexion larvae [8.6 mm BL (day 19; Fig. 4b). Maximum
Table 1 Body length (mm) and age in days at each developmental BD initially 2833% BL (Fig. 4c), proportion rapidly
stage of Trichogaster trichopterus
decreasing to 1618% BL with yolk absorption and body
Stage BL (mm) Age (days) Number of fish extension in flexion larvae \5.2 mm BL (day 9; Fig. 4c),
Preflexion larva 2.44.6 011 39
subsequently increasing continuously to over 40% BL in
Flexion larva 4.36.7 713 9
juveniles (Fig. 4c). ED initially 1012% BL (Figs. 2a, 3d),
Postflexion larva 5.911.9 1330 23
proportion rapidly decreasing to 89% with head and body
extension in preflexion larvae \3.4 mm BL (day 1;
Juvenile [11.9 [30 17
Figs. 2b, 3d), increasing subsequently to 1113% BL in

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Fig. 2 Laboratory-reared three-spot gourami Trichogaster trichopte-
rus. a Newly hatched larva [2.5 mm BL, MTUF-P(L)-26591];
b preflexion larva, day 1 [3.1 mm BL, MTUF-P(L)-26592]; c pre-
flexion larva, day 2 [3.4 mm BL, MTUF-P(L)-26593]; d preflexion
larva, day 3 [3.7 mm BL, MTUF-P(L)-26594]; e preflexion larva, day
5 [4.0 mm BL, MTUF-P(L)-26595]; f preflexion larva, day 7 [4.2 mm
postflexion larvae \7.2 mm BL (day 22; Fig. 3d) and
decreasing slightly thereafter. SnL 0.51.1% BL on day 1,
increasing to 79% BL in postflexion larvae\10.2 mm BL
(day 25), and became constant thereafter (Fig. 4e). UJL
23% BL on day 1, increasing to 710% BL in juveniles
\17.7 mm BL, subsequently decreasing slightly to 68%
(Fig. 4f). First soft ray of pelvic fin elongating following
123
S. Morioka et al.
BL, MTUF-P(L)-26596]; g preflexion larva, day 9 [4.4 mm BL,
MTUF-P(L)-26597]; h flexion larva, day 11 [5.5 mm BL, MTUF-
P(L)-26598]; i postflexion larva, day 13 [6.7 mm BL, MTUF-P(L)-
26599]; j postflexion larva, day 19 [8.6 mm BL, MTUF-P(L)-26600].
a0 , b0 , c0 , d0 , e0 , f0 , g0 anterior dorsal view. YS yolk sac, GB gas bladder
initial appearance, proportion attaining over 70% BL by
day 35 (Fig. 4g).
Fin development. Dorsal soft rays initially appearing in
postflexion larvae at 6.5 mm BL and in all postflexion
larvae [6.9 mm BL (day 1316; Figs. 2i, 5a), spines
appearing initially in postflexion larvae at 7.1 mm BL and
in all postflexion larvae [8.6 mm BL (day 19), attaining
Morphological development of Trichogaster trichopterus
Fig. 3 Laboratory-reared (ac) and wild (df) three-spot Trichogas-
ter trichopterus. a Postflexion larva, day 25 [10.6 mm BL, MTUF-
P(L)-26610]; b juvenile, day 40 [17.3 mm BL, MTUF-P(L)-24715];
full complement (VIVII, 910) in postflexion larvae
[10.6 mm BL (day 30; Fig. 5a); soft ray segmentation ini-
tiated in postflexion larvae [7.8 mm BL (day 1619),
completed (89 segmented rays) in postflexion larvae
[8.6 mm BL (day 1922). Anal soft rays appearing in all
flexion and postflexion larvae [6.5 mm BL (day 1316;
Figs. 2i, 5b), spines initially appearing in postflexion larvae
at 7.1 mm BL and in all postflexion larvae[8.6 mm BL (day
19), attaining full complement (VIIIX, 3234) in postflex-
ion larvae [11.0 mm BL (day 22; Fig. 5b); soft ray seg-
mentation initiated in postflexion larvae [7.1 mm BL (day
19), completed (3233 segmented rays) in juveniles. Caudal
soft rays initially appearing in flexion larvae at 4.3 mm BL
and in all flexion larvae [4.6 mm BL (day 711; Figs. 2g,
5c), attaining full complement (9 ? 9 = 18) in postflexion
larvae [7.2 mm BL (day 1922; Figs. 2j, 5c); soft ray seg-
mentation initiated in flexion larvae[5.4 mm BL (day 13),
completed (16 segmented rays) in postflexion larvae
[8.6 mm BL (day 19). Pectoral soft rays initially appearing
in postflexion larvae at 7.1 mm BL and in all postflexion
larvae[8.6 mm BL (day 1619; Figs. 2j, 5d), attaining full
complement (1011) in postflexion larvae[8.6 mm BL (day
2530; Figs. 3a, 5d); soft ray segmentation initiated in
postflexion larvae [7.1 mm BL (day 19), completed (89
segmented rays) in juveniles [9.2 mm BL (day 2530).
Pelvic soft rays initially appearing in postflexion larvae at
57
c juvenile, day 40 [25.5 mm BL, MTUF-P(L)-24716]; d postflexion
larva [11.6 mm BL, MTUF-P(L)-24717]; e juvenile [17.4 mm BL,
MTUF-P(L)-24718]; f juvenile [24.5 mm BL, MTUF-P(L)-24720]
7.2 mm BL and in all postflexion larvae [8.6 mm BL (day
1922; Figs. 2j, 5e), a spine appearing in postflexion larvae
[9.2 mm BL (day 25), attaining full complement (I, 4) in
juveniles[11.9 mm BL (day 30; Figs. 3b, 5e); first soft ray
becoming remarkably elongate following its appearance at
7.2 mm BL (day 22; Fig. 3b, c); first soft ray segmentation
initiating in postflexion larvae[10.1 mm BL (day 25), other
soft rays not segmented. Branched rays not present in any
fins.
Pigmentation. Light punctate and stellate melanophore
deposition in eyes observed in newly hatched larvae (day 0;
Fig. 2a), densely deposited thereafter (Figs. 2bj, 3ac).
Many light stellate melanophores initially sparsely over
surface of yolk sac in newly hatched larvae, decreasing in
number with yolk absorption (Fig. 2ag). Light melano-
phore present in interorbital and occipital regions in day 0
larvae (Fig. 2a), thereafter deposited with increasing den-
sity with growth and covering entire head (Figs. 2bj, 3a
c); several punctuate melanophores appearing on upper jaw
in preflexion larvae at 3.4 mm BL (day 2; Fig. 2c), on lower
jaw in preflexion larvae at 3.7 mm BL (day 3; Fig. 2d)
increasing in number with growth (Figs. 2dj, 3ac).
Many punctate melanophores present on the throat and
ventral region of abdomen in day 01 larvae (Fig. 2a, b),
decreasing and completely lost in postflexion larvae at
6.7 mm BL (day 13). A few stellate melanophores
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Fig. 4 Proportions of a head

length (HL), b preanal length
a e
(PAL), c body depth (BD), d eye
diameter (ED), e snout length
(SnL), f upper jaw length (UJL),
and g first soft ray length of
pelvic fin (P2FSR) to body
length (BD) in laboratory-reared
(solid circles) and wild (open
circles) larvae and juvenile
three-spot gourami Trichogaster b f
trichopterus

c g

appearing on operculum in day 12 larvae (Fig. 2b, c),
those in operculum upper region increasing, lower region
disappearing with growth (Figs. 2dj, 3ac).
Melanophores on lateral body surface initially few
(Fig. 2a), subsequently over a dozen punctate melano-
phores appearing on lateral line following its appearance in
day 1 larvae (Fig. 2b), increasing with growth, but not
visible in postflexion larvae at 10.6 mm BL (day 25;
Fig. 3a); several punctate/stellate melanophores sparsely
distributed on lateral body surface in postflexion larvae at
6.7 mm BL (day 13; Fig. 2i) in addition to those on lateral
line, subsequently becoming punctuate and covering most
of upper lateral body in postflexion larvae at 8.6 mm BL
(day 19; Fig. 2j); punctate melanophores appearing
beneath lateral line in postflexion larvae at 6.7 mm BL
(day 13; Fig. 2i), increasing and forming 35 light hori-
zontal bands on posterolateral half of the body in
postflexion larvae and juveniles \10.6 mm BL (Fig. 3a);
subsequently, the band distributions became irregular but
more distinct, the bands formed on caudal fin in juvenile
[ca. 20 mm BL (Fig. 3c); several un-pigmented oblique
slender patches appearing on anterodorsal region in juve-
niles [12.0 mm BL (Fig. 3b), becoming more distinct
thereafter, but less distinctive in juveniles [20 mm BL
(Fig. 3c); dozens of punctate melanophores appearing
around caudal margin of the body in postflexion larvae at
6.7 mm BL (day 13; Fig. 2i), becoming more dense and
forming a large dark spot with growth (Figs. 2j, 3ac);
dozens of punctate melanophores appearing on the center
of lateral body in postflexion larvae [8.6 mm BL (Fig. 2j),
becoming more dense and forming a large dark spot with
growth (Fig. 3ac).
Dozens of small punctate melanophores present on body
along dorsal fin fold except posterior tip in day 1 larvae,

123
Morphological development of Trichogaster trichopterus
a present on body along ventral fin fold in day 1 larvae
b lateral body (Fig. 3c).
c phores appear on dorsal and anal fins in postflexion larvae
d BL (day 19) as primordial hypertrophy of the upper portion
e seven proportional characters examined here, similar trends
Fig. 5 Relationships between body length (mm) and fin ray numbers
in laboratory-reared (solid circles) and wild (open circles) larval and
juvenile three-spot gourami Trichogaster trichopterus. a Dorsal fin
(DFR); b anal fin (AFR); c caudal fin (CFR); d pectoral fin (P1FR);
e pelvic fin (P2FR)
subsequently increasing in number and covering entire
dorsal region of body in postflexion larvae [8.6 mm BL
from day 19 (Fig. 2i). Small punctate melanophores
59
(Fig. 2b), subsequently on anal fin pterygiophores with
melanophores developing distally in postflexion larvae
[6.7 mm BL (day 13; Fig. 2i). Melanophores posterior to
anus constant along base of pterygiophores until postflex-
ion larval stage (Fig. 3a), subsequently increasing and
covering most of anal fin ventral margin (Fig. 3b), but un-
pigmented area appearing along anal fin base in juveniles
[20 mm BL thereafter as horizontal band development on
Small dense melanophores present on the dorsal surface
of the gas bladder from its initial appearance in day 9
larvae (Fig. 2g), disappearing as the gas bladder becomes
invisible in postflexion larvae at 8.6 mm BL (day 19;
Fig. 2j). Several punctate melanophores appear on the
caudal fin in preflexion larvae at 4.4 mm BL (day 9;
Fig. 2g), increasing thereafter; several punctate melano-
at 6.7 mm BL (day 13; Fig. 2i), with those on the lower
half of the anal fin becoming more dense in postflexion
larvae at 10.6 mm BL (day 25; Fig. 3a), with the density
subsequently increasing as melanophores cover the entire
anal fin in juveniles (Fig. 3b, c).
Air-breathing and labyrinth organ development. Air-
breathing was observed on days 1820 after hatching. The
labyrinth organ appeared in postflexion larvae [7.9 mm
of first gill arch (Fig. 6b), with hypertrophy subsequently
developing (Fig. 6c, d) and forming a spherical structure
on the upper gill arch in postflexion larva at 11.6 mm BL
(day 30; Fig. 6e), a fan-shaped membranous structure in
juveniles at 15.1 mm BL (day 30; Fig. 6f) and a mem-
branous, eventually double-layered structure in juveniles at
22.9 mm BL (day 40; Fig. 6g).
Comparison between reared and wild specimens. Of
were observed in five characters between the laboratory-
reared and wild specimens, i.e., HL, PAL, maximum BD,
SnL and UJL (Fig. 4). However, proportions of ED and the
first soft ray of the pelvic fin in the laboratory-reared
juveniles of ca. 1825 mm BL size range (912 and
7178%) tended to be smaller than those in seven wild
ones (18.524.5 mm BL, 1113 and 8490%, respectively)
(Fig. 5d, g). Pigmentation patterns and numbers of fin rays
in the reared specimens were not distinctively different
from those in the wild specimens under the size range of
the latter (11.624.5 mm BL; Figs. 3, 5).
Discussion
The developmental stages of Trichogaster trichopterus
were determined in this study, the species being considered
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Fig. 6 Development of labyrinth organ in laboratory-reared larval
and juvenile three-spot gourami Trichogaster trichopterus. a Flexion
larva, day 16 [6.6 mm BL, MTUF-P(L)-26603]; b postflexion larvae,
day 19 [7.9 mm BL, MTUF-P(L)-26604]; c postflexion larva, day 22
[9.7 mm BL, MTUF-P(L)-26605]; d postflexion larva, day 22
to have attained the juvenile stage by 11.9 mm BL (based
on completion of aggregate fin ray numbers; Table 1;
Figs. 3b, 5). However, body proportions of laboratory-
reared fish did not become constant (max. 26.1 mm BL;
Fig. 4). Comparison between laboratory-reared and wild
specimens showed the proportions of ED and the first soft
ray length of the pelvic fin being smaller in the former than
in the latter in the size range of ca. 1825 mm BL (Fig. 4).
Such morphometric differences between laboratory-reared
and wild fish, being often observed in other fishes, e.g.,
Pagrus major (see Matsumiya and Kanamaru 1987) and
Thunnus albacares (see Shimizu and Shiozawa 2004), may
be attributable to differences in diets and water temperature
(Matsumiya and Kanamaru 1987), although further exam-
inations are required.
The 10-day yolk sac period observed in the present
study (Fig. 2) indicated that Trichogaster trichopterus has
a ca. 8-day preparatory period from days 3 (start of feed-
ing) to 10 (just before complete yolk absorption) inclusive
for the shift from endogenous to exogenous energy-
dependent periods (under the experimental temperature
regime during this study). The shift from endogenous to
exogenous energy-dependent periods has been examined in
several species of marine fishes, covering aspects of their
development and survival during the larval stages (Kohno
1998; Moteki et al. 2001), and similar investigations of
freshwater fishes have recently been reported for several
species, i.e., the North African catfish Clarias gariepinus
(Clariidae; Matsumoto et al. 2001), the climbing perch
Anabas testudineus (Anabantidae; Morioka et al. 2009), the
snakeskin gourami Trichogaster pectoralis (Os-
phronemmidae; Morioka et al. 2010a), the sutchi catfish
Pangasianodon hypophthalmus (Pangasiidae; Morioka
et al. 2010b), the goldfin tinfoil barb Hypsibarbus malcolmi
(Cyprinidae; Ogata et al. 2010) and Hemibagrus filamentus
(Bagridae; Morioka and Vongvichith 2011), including
123
S. Morioka et al.
[10.8 mm BL, MTUF-P(L)-26606]; e postflexion larva, day 30
[11.6 mm BL, MTUF-P(L)-26607]; f juvenile, day 30 [15.1 mm BL,
MTUF-P(L)-26608]; g juvenile, day 40 [22.9 mm BL, MTUF-P(L)-
26609]. LO labyrinth organ, MS membranous structure(s), GR gill
rakers. Bar 1 mm
survival strategies during early life stages as well as any
improvements in seed productivity. By comparison,
T. pectoralis, a congener of T. trichopterus, showed a ca.
10-day preparatory period from days 2 to 11 inclusive
(Morioka et al. 2010a) under similar water temperatures, a
period mostly coinciding with that of T. trichopterus in the
present study. However, the other aforementioned species
showed much shorter preparatory periods, i.e., 4 days in
Clarias gariepinus, 5 days in Anabas testudineus, 1 day in
Pangasianodon hypophthalmus, 0 days in Hypsibarbus
malcolmi and 3 days in Hemibagrus filamentus. These
longer preparatory periods in the two osphronemids
examined here, allowing a slower shift from endogenous to
exogenous energy intake, may be attributable to the
uniqueness of the reproductive strategies of these two
species, i.e., eggs spawned in the bubble nest with parental
care during egg and larval stages (Kramer 1973; Ruber
et al. 2006), although confirmation is required.
Although the function of the labyrinth organ and asso-
ciated ecological aspects (Burggren and Haswell 1979)
have been reported for adult T. trichopterus and its con-
gener T. pectoralis (see Burggren 1979), the ontogeny and
developmental morphology of the former in larval and
juvenile stages are poorly understood. In the air-breathing
osphronemid Macropodus opercularis (see Matayoshi and
Shokita 1982), anabantid A. testudineus (see Morioka et al.
2009) and T. pectoralis (see Morioka et al. 2010a),
descriptions of the developmental morphology of the lab-
yrinth organs showed that the occurrence of air-breathing
followed the onset of primordial hypertrophy of the first
gill arch and continued with subsequent enlargement of the
organ. In the present study, air breathing was observed
since days 1820 after hatching, this coinciding with the
appearance of primordial hypertrophy of the upper portion
of the first gill arch (Fig. 6b). Since T. trichopterus is an
obligate air-breather (Burggren and Haswell 1979), it is
Morphological development of Trichogaster trichopterus
highly resistant to low levels of dissolved oxygen in the
surrounding water, suggesting a considerable aptitude of
the species for seed production and aquaculture under high
stocking densities. In this study, the labyrinth organ of
T. trichopterus was noted to possess only a single-layered
membrane in 15.1 mm BL juveniles and a two-layered
membrane in a 22.9 mm BL juveniles (Fig. 6), whereas it
was more developed at 14.9 mm BL (having three-layered
membranes) in A. testudineus (see Morioka et al. 2009).
This difference, as well as the faster morphological
development in A. testudineus (see Morioka et al. 2009),
may be related to specific survival strategies during early
life stages, although more information is necessary.
Although nine species of Osphronemidae, including
T. trichopterus, are known to be distributed in Laos (Kottelat
2001), their early life stage morphology has not been
described to date, except for a congener of the latter,
T. pectoralis (see Morioka et al. 2010a). Hence, the mor-
phology of larvae and juveniles of both species is com-
pared, being valuable in cases of in situ species
identification. Although the size of newly hatched
T. trichopterus larvae (2.5 0.1 mm BL, ranging from 2.4 to
2.5 mm BL) were slightly larger than those of T. pectoralis
(2.2 0.1 mm BL, ranging from 2.1 to 2.3 mm BL), the
body shape and melanophore distribution were similar
during the preflexion larval stage, the duration of the yolk
sac stage also being similar (11 days for the former,
12 days for the latter). However, a difference in myomere
numbers became apparent during late preflexion (ca. 5 mm
BL) and flexion larval stages (46 ? 2426 = 2931 in
the former, 78 ? 2526 = 3234 in the latter). A large
dark spot on the caudal margin of the body in both species
formed during the flexion larval stage at similar sizes
([6.7 mm BL in T. trichopterus, [6.1 mm in T. pecto-
ralis). However, a second large spot posteriorly on the
center of lateral body in the former ([8.6 mm BL) was
absent in the latter. In addition, the PAL was slightly
greater in the former in the juvenile stage (4952%, com-
pared with 4550% in the latter), the maximum BD was
greater in the former following the postflexion larval stage
(reaching[40% in the juvenile stage, ca. 30% in the latter),
and the first pelvic fin soft ray length was much greater in
the former ([50%, reaching [70% in the juvenile stage
and 3040% in individuals of similar size range in the
latter).
Acknowledgments We express our sincere gratitude to K. Sano,
Fisheries and Aquaculture International Co., Ltd., and the staff of the
Aquaculture Improvement and Extension Project of the Japan Inter-
national Cooperation Agency for their kind cooperation on brood-
stock collection. We are also grateful to L. Khamsivilay, Living
Aquatic Resources Research Center, Laos, for his logistical support.
Our thanks also go to G. Hardy for his polite and constructive English
revision.
61
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