Aquaculture 256 (2006) 489 – 501

www.elsevier.com/locate/aqua-online

Ontogenetic development of the digestive system in yellowtail

kingfish Seriola lalandi larvae
Ben Nan Chen a,1 , Jian G. Qin a,⁎, Martin S. Kumar b ,
Wayne Hutchinson b , Steven Clarke b
a
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia
b
South Australian Research and Development Institute, PO Box 120, Henley Beach, SA 5022, Australia
Received 16 March 2005; received in revised form 22 November 2005; accepted 27 January 2006

Abstract

Ontogenetic development of the digestive tract and associated organs in yellowtail kingfish (Seriola lalandi, Family:
Carangidae) larvae was morphologically and histologically examined using light microscopy from hatching to 36 days after hatch
(DAH). The first developmental phase started from hatching when the digestive tract was a simple tube and ended with the onset of
exogenous feeding. The second developmental phase was from the start of exogenous feeding to the appearance of gastric glands,
in which eosinophilic supranuclear vacuoles occurred in the hindgut on 4 DAH and lipid vacuoles occurred in the anterior midgut
on 5 DAH, indicating the start of protein and lipid absorption. After the stomach formation on 5 DAH, the digestive tract was
distinctively divided into buccopharyngeal cavity, oesophagus, stomach, midgut and hindgut. Following the intestinal curve on 8
DAH, goblet cells, pharyngeal teeth, taste buds and the tongue also appeared. The third developmental phase started from the
appearance of gastric glands on 15 DAH and continued onward. The stomach was divided into cardiac, fundic and pyloric regions
when the pyloric caeca formed on 18 DAH. Gastric glands distributed in cardiac and fundic regions, but not in the pyloric region.
The formation of the fundic stomach signalled the starting point of weaning. This study shows the quick development of the
digestive system in yellowtail kingfish, and the results should lead to a better understanding of the ontogeny of fast-growing fish
larvae and improvement of larval rearing success in hatcheries.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Ontogeny; Histology; Digestive system; Morphology; Weaning; Yellowtail kingfish; Seriola lalandi

1. Introduction an efficient digestive system enables fish to capture,

Successful development of the digestive system is
crucial for the survival and growth in fish larvae because
⁎ Corresponding author. Tel.: +61 8 8201 3045; fax: +61 8 8201
3015.
E-mail address: jian.qin@flinders.edu.au (J.G. Qin).
1
Present address: South Australian Research and Development
Institute, PO Box 120, Henley Beach SA 5022, Australia.
0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2006.01.041
ingest, digest and absorb food (Kjorsvik et al., 2004).
Although larval fish may be morphologically capable of
capturing food items at first feeding (Segner et al., 1994;
Bisbal and Bengtson, 1995), the digestive system needs
a series of developmental changes before being fully
functional (Govoni et al., 1986; Canino and Bailey,
1995). Knowledge on the structural development of the
digestive system is essential to understand the digestive
physiology and determine the appropriate timing to
490 B.N. Chen et al. / Aquaculture 256 (2006) 489–501
wean fish larvae (Watanabe and Kiron, 1994; Baglole et
al., 1997; Cahu and Zambonino Infante, 2001).
Ontogenetic development of the digestive system in
most teleost fish is generally divided into three major
phases (Buddington, 1985; Boulhic and Gabaudan,
1992; Bisbal and Bengtson, 1995). The first phase starts
from hatching and ends at the completion of endogenous
feeding. During this period, larvae depend on energy
reserves in the yolk sac and oil globules. Toward the end
of the first phase, fish experience a transition of
endogenous to exogenous feeding before exclusively
feeding on external food. The second phase starts from
the onset of exogenous feeding and ends before the
formation of gastric glands in the stomach, characterized
by the lack of sufficient digestive capabilities (Budding-
ton, 1985; Boulhic and Gabaudan, 1992). During this
phase, larval fish mainly depend on pinocytosis and
intracellular digestion and absorption (Watanabe, 1982).
As a result, fish larvae usually feed on live food such as
rotifers that can be easily ingested and digested. The third
phase starts from the presence of gastric glands and
pyloric caeca to metamorphosis onward, indicating the
functional maturation of the digestive system (Tanaka,
1971; Bisbal and Bengtson, 1995). The third develop-
mental phase coincides with metamorphosis, when the
digestive system anatomically and physiologically is
ready to accept artificial pellets (Bisbal and Bengtson,
1995; Gordon and Hecht, 2002). Despite the overall
similarity of the developmental pattern, the duration of
each developmental phase varies among fish species.
The understanding of ontogenetic development of the
digestive system is crucial for larval fish rearing in any
economically important aquaculture species.
Fragmental information exists on the histological
development of the digestive system of yellowtail
kingfish Seriola lalandi (Umeda et al., 1987) and
another yellowtail species Seriola quinqueradiata in the
same genus (Umeda and Ochiai, 1973, 1975). Umeda et
al. (1987) found that the liver became functional before
the onset of exogenous feeding in yellowtail kingfish.
These authors further noticed that digestion and
absorption occurred 3–4 days after the onset of
exogenous feeding as evidenced by the lipid droplets
and supranuclear vacuoles in the intestine enterocytes.
However, the information about its ontogenetic devel-
opment of the digestive system is incomplete comparing
to some other important commercial species such as
turbot Scophthalmus maximus L. (Cousin and Laur-
encin, 1987; Hoehne-Reitan et al., 2003), gilthead
seabream Sparus aurata L. (Sarasquete et al., 1995;
Calzada et al., 1998), Senegal sole Solea senegalensis
(Ribeiro et al., 1999; Arellano et al., 2001) and sea bass
Dicentrarchus labrax L. (Cahu and Zamboninon
Infante, 1995; Peres et al., 1998).
Yellowtail kingfish has been used for commercial
aquaculture in South Australia for its fast growth, high
flesh quality and suitability for cage culture (Fowler et al.,
2003). Although the life cycle is closed in yellowtail
kingfish (Tachihara et al., 1997; Gillanders et al., 1999;
Poortenaar et al., 2001), high larval deformities and
mortalities have hindered the industry development.
Therefore, the study of larval fish nutritional physiology
in this species should provide information to solve some
of these problems (Govoni et al., 1986; Segner et al.,
1993). However, the study of histological development of
the larval fish digestive system should be undertaken as a
first step because most physiological functions are based
on the organ development (Hamlin et al., 2000; Govoni,
2004). The objective of this study was therefore to
examine the structural changes during the ontogeny of the
digestive system of hatchery-reared yellowtail kingfish
from hatching until metamorphosis, aiming to under-
stand the sequence of organ development and provide
fundamental knowledge on hatchery management for
fast-growing species such as yellowtail kingfish.
2. Materials and methods
2.1. Eggs and larval fish rearing
Fertilized eggs of the same batch of brood yellowtail
kingfish were obtained from the Arno Bay, South
Australia, and transported to the South Australian
Aquatic Sciences Centre, Adelaide. Upon arrival, all
eggs were hatched in 120-L fibreglass incubators at
21°C. After hatching, the larvae were stocked in 600-L
fibreglass rearing tanks at 60 fish/L. All rearing tanks
were supplied with filtered seawater with a 5-μm filter in
a flow-through system with a water exchange rate of
1.2–3.0 L/min. Two air stones were used in each tank to
maintain dissolved oxygen at saturation and also to
promote a homogeneous distribution of microalgae and
live foods (rotifers and Artemia nauplii). Light intensity
at 3000–6000lx and a photoperiod of 13h light and 11 h
dark were provided. Salinity was maintained at 38‰
throughout the experiment and water temperature was
controlled at 24 ± 1.3°C.
Rotifers (Brachionus plicatilis) were given to fish
larvae on 2days after hatching (DAH) until 12 DAH at
10 individuals/mL. The rotifers fed with microalgae
(Nannochloropsis oculata) were enriched with DHA
Selco (INVE Aquaculture) for 12 h before being added
into the fish tanks. A microalga (Isochrysis sp.) was also
added to the larval fish tanks as food for the rotifers.
 B.N. Chen et al. / Aquaculture 256 (2006) 489–501
Artemia nauplii enriched with dried algae (Bio-Marine
Algamac 3050™) were introduced to the rearing tanks
from 10 to 24 DAH at 5 individuals/mL. Commercial
pellet feed (Proton 3, INVE Aquaculture) was intro-
duced to the fish from 18 DAH to 36 DAH to the end of
the experiment (36 DAH).
2.2. Fish sampling and growth measurements
The fish larvae were randomly collected in triplicate
from the rearing tanks each day from hatching to 5 DAH,
and then specimens were collected on 8, 12, 15, 18, 24
and 36 DAH to examine the development of the
digestive system. Standard length was measured, as a
mean of 10 fish, from the upper jaw to the end of the
notochord for larvae, or to the end of the vertebral
column for juveniles, under a microscope to the nearest
0.05 mm on each sampling day. The larval growth was
determined by the measurement of the absolute growth
rate (AGR) as mm/day and specific growth rate (SGR) as
%/day (Hopkins, 1992). AGR was calculated as: AGR =
(SLf − SLi) / Δt, and SGR was determined as: SGR = 100
(LnSLf − LnSLi) / Δt, where SLf and SLi are the final and
initial fish standard length (mm), respectively, and Δt is
the time interval (days) between samples.
2.3. Morphological observation
Fifteen fish larvae from each of three replicate tanks
were collected on each sampling day and 5 fish was
randomly examined for the morphology of the digestive
tract under a Leica dissecting microscope and a Nikon
microscope. From 0 to 12 DAH, since fish were transparent,
all larvae were directly observed under the microscope after
being anaesthetized in Benzocaine solution. However, from
15 DAH onward, since the body wall became opaque, the
digestive tract was dissected out for observation. Photo-
graphs were taken with a Canon digital photomicrographic
attachment during the observation.
2.4. Histological analysis procedures
On each sampling day, 30 larvae from each of three
replicate tanks were fixed in Zamboni's fixative after
being anaesthetized in Benzocaine solution. The fixed
fish were individually embedded in paraffin blocks and
sectioned in serial sagittal sections (5-μm thick) using a
Leica RM 2135 rotary microtome. The haematoxylin-
eosin (HE) stain was used for general histological
observations. Masson trichrome was used to stain
collagen, while the periodic acid-Schiff technique
(PAS) was used to stain neutral mucins (Pearse, 1985;
491
Bancroft and Gamble, 2002). The slide with sections
was mounted permanently using DePex. The sections of
five fish were randomly examined under an Olympus
light microscope. Photographs were taken with a Canon
digital photomicrographic attachment.
3. Results
3.1. Fish growth and general development of the
digestive system
Standard length of larval fish averaged 4.30 mm at
hatching (0 DAH), and increased to 22.56 mm on 36
DAH (Fig. 1). The absolute growth rate and specific
growth rate under laboratory conditions were 0.51 mm/
day and 4.60%/day, respectively. Upon hatching, the fish
digestive tract was a straight tube lying dorsally to the
yolk sac with posterior portion bended ventrally (Fig.
2a). The lumen was observed in the posterior of the tube
and joined the urine bladder at the posterior intestine
(Fig. 2b). This connection was further confirmed by the
observation of liquid discharge from the urine bladder
into the hindgut cavity. Although the anus was open, the
mouth was closed on 0 DAH (Fig. 2a,b). There was no
sign of incipient liver and pancreas at this stage. Rotifers
were supplied to the fish on 2 DAH. However, we did
not find any rotifer in the fish gut on that day. Rotifers
were first detected in the fish gut on 3 DAH through the
gut analysis. On 4 DAH, food particles were easily
identified in the midgut (Fig. 2d). Since most oil
globules in the yolk sac depleted by 8 DAH, the mixed
feeding should have occurred between 3 and 8 DAH.
Although the growth of larvae was not significant during
4–8 DAH, mixed feeding should be critical for the
development of fish organ during this period.
After mouth opening on 2 DAH, the digestive tract
elongated and the gut lumen increased in size (Fig. 2c).
The formation of the intestinal valve on 4 DAH divided
the incipient intestine into midgut and hindgut (Fig. 2d).
At this stage, the amount of yolk sac and oil globules
were reduced, but remained in the anterior abdominal
cavity. The liver was behind the yolk sac. When the gut
curve formed on 8 DAH at the anterior midgut, the size
of digestive tract substantially increased (Fig. 2e). The
observation of pyloric caeca on 24 DAH marked the last
major morphological change in the digestive tract
development (Fig. 2f).
3.2. Buccopharynx
Buccopharyngeal cavity was not formed on 0 DAH
(Fig. 3a), but oral valves seen as dorsal and ventral
492
25
Phase
I II
20
Standard length (mm)
15
10
5 and
Yolk
0
012
Fig. 1. Standard length of yellowtail kingfish from 0 to 36days after hatching. Developmental phases are shown as Phases I, II and III, and diet types
are shown below the growth curve.
epithelial folds were observed on 1 DAH (Fig. 3b).
The buccopharyngeal cavity lined by a single layer of
squamous epithelium was observed on 1 DAH before
mouth opening on 2 DAH. On 4 DAH, a stratified
squamous epithelium with connective tissues formed
the buccopharyngeal mucosa (Fig. 3c).
Pharyngeal tooth occurred on 8 DAH in the
pharynx (Fig. 3d), and numerically increased as fish
grew. Taste buds also appeared in the posterior of the
buccopharyngeal cavity on 8 DAH (Fig. 3d) and
became more numerous on 15 DAH (Fig. 4c). The
tongue formed on 8 DAH by a simple thickening of
the oral floor consisting of a stratified squamous
epithelium (Fig. 3d).
3.3. Oesophagus
Upon hatching, the oesophagus appeared as a simple
tube similar to the incipient gut, and was lined by a
single layer of simple cuboidal epithelium (Fig. 4a).
Before the formation of the buccopharyngeal cavity, the
digestive tract was closed at the oesophagus. On 3 DAH,
the oesophageal lumen lined with a stratified squamous
epithelium was formed to connect the buccopharyngeal
cavity and intestine (Fig. 4b). On 4 DAH, the posterior
oesophagus expanded to form the stomach (Fig. 3c).
The oesophagus was surrounded by a circular layer of
striated muscles, and the muscle layer became thicker as
larvae grew.
B.N. Chen et al. / Aquaculture 256 (2006) 489–501
Phase Phase
III
Rotifers and
Artemia nauplii
Artemia
nauplii Compound diet
Artemia compound
Rotifers nauplii diet
(Weaning)
4 5 8 12 15 18 24 36
Days after hatching
At first feeding on 3 DAH, no mucous cells and folds
were observed in the oesophagus. However, on 5 DAH,
mucous cells were formed in the oesophagus mucosae
and rapidly increased in number, coinciding with fish
feeding on rotifers at this stage. The mucous cells were
stained in red by PAS on 12 DAH, indicating the
presence of neutral glycoproteins or mucosubstances.
Numerous mucous cells were found in the posterior
oesophagus on 15 DAH when food was changed from
rotifers to Artemia nauplii (Fig. 4c, d).
Two days after exogenous feeding on 5 DAH,
longitudinal folds formed along the oesophagus, and
became abundant on 8 DAH (Fig. 5b). Oesophagus
mucosal folds formed by a simple cubic epithelium.
More longitudinal folds and mucous cells formed
between the oesophagus and stomach on 15 DAH
(Fig. 4d), but there were no substantial histological
changes from 15 to 36 DAH.
3.4. Stomach
The presumptive stomach appeared as a bulge at
the end of the oesophagus on 4 DAH (Fig. 3c). On 5
DAH, a preliminary pyloric sphincter developed and
the mucosae of stomach formed by a simple cubic
epithelium similar to that in oesophageal mucosae
(Fig. 5a). On 8 DAH, the stomach became more
elongated and the border between the stomach and
oesophagus was differentiated by an abrupt change
 B.N. Chen et al. / Aquaculture 256 (2006) 489–501 493

Fig. 2. Morphological observations of the digestive tract during the development of yellowtail kingfish larvae. (a) General view of the newly hatched
larva on 0day after hatching (DAH). (b) Posterior region of the incipient intestine of newly hatched larvae with a narrow lumen (400×). Note the anus
opening, and the connection between the urine bladder and incipient intestine (arrowhead). (c) View of the digestive tract on 2 DAH larvae. Note the
mouth opening (arrowhead), elongated incipient intestine and unpigmented eye. (d) Incipient intestine on 4 DAH. Note the intestinal valve, midgut,
hindgut and food particles (arrowheads). (e) Curved gut on 8 DAH. (f) Pyloric caeca (arrows) between the pyloric stomach and anterior midgut on 24
DAH. Abbreviations: AN, anus; E, eye; HG, hindgut; IN, incipient intestine; IV, intestinal valve; LU, lumen; MG, midgut; OE, oesophagus; OG, oil
globule; ST, stomach; UB, urine bladder; YS, yolk sac.

from squamous epithelium to a simple cuboidal
epithelium (Fig. 5b). On 12 DAH, the pyloric sphincter
separated the stomach from the midgut and the
stomach then comprising the cardiac and pyloric re-
gions (Fig. 5c).
On 15 DAH, the presence of gastric glands advanced
the presumptive stomach (Fig. 5d), coinciding with fish
feeding on Artemia nauplii. The gastric glands were
simple acinus glands beneath the epithelium between
the cardiac and pyloric regions (Fig. 5d), and the gastric
glands were composed of single-type secretory cells and
PAS-positive apical borders. On 18 DAH, the stomach
was further divided into cardiac, fundic and pyloric
regions (Fig. 5e). The gastric glands scattered in the
cardiac and fundic regions but not in the pyloric region
(Fig. 5f). The fundic region was elongated as larvae
grew and formed the largest portion of the stomach. By
36 DAH no goblet cells were observed in the epithelium
in the stomach.
3.5. Intestine and pyloric caeca
The incipient intestine of a newly-hatched larva was
lined by a single layer of columnar cells (Fig. 6a).
Structural changes of the larval intestine occurred
between 3 and 4 DAH when fish shifted from
endogenous nutrition to a mixture of endogenous and
exogenous nutrition. On 4 DAH, the intestinal valve
divided the intestine into midgut and hindgut (Fig. 6b).
Subsequently, numerous acidophilic supranuclear
vacuoles appeared in the hindgut, indicating the start
of protein digestion and absorption in the gut (Fig. 6b).
On 5 DAH, lipid vacuoles appeared in the enter-
ocytes of the anterior midgut (Fig. 6c), but not in the
494 B.N. Chen et al. / Aquaculture 256 (2006) 489–501

Fig. 3. Sagittal section of the buccopharyngeal cavity during the development of yellowtail kingfish larvae. (a) Head of a newly hatched larva. Note
the oil globule in front of the yolk sac; the asterisk shows the absence of oral valve; HE. (b) Larva of 1day after hatching (DAH), showing the
buccopharyngeal cavity and oral valve; HE. (c) 4 DAH larva, showing the buccopharyngeal cavity. Note the presumptive stomach derived from the
oesophagus expansion indicated by an asterisk; HE. (d) Tongue appeared on 8 DAH showing pharyngeal teeth (arrows) and taste buds (arrowheads);
HE. Abbreviations: BC, buccopharyngeal cavity; E, eye; HE, haematoxylin-eosin; L, liver; MF, muscular fibres; N, notochord; OE, oesophagus; OG,
oil globule; OV, oral valve; T, tongue; YS, yolk sac.

Fig. 4. Sagittal section of the oesophagus in yellowtail kingfish during ontogeny. (a) Oesophagus in a newly hatched larva; HE. (b) Oesophagus on
3days after hatching (DAH). Note the oesophagus (arrowhead) lined with a stratified squamous epithelium, hepatocyte cells in the liver and zymogen
granules in the pancreas, and pancreatic duct (arrow); HE. (c) Oesophagus in larva on 15 DAH. Note the pharyngeal tooth (arrow), taste buds
(arrowheads) and mucous cells in the oesophagus (asterisk); HE. (d) Mucous cells (arrowheads) at the end of oesophagus on 15 DAH; PAS.
Abbreviations: BC, buccopharyngeal cavity; E, eye; H, heart; HE, haematoxylin-eosin; IN, incipient intestine; K, kidney; L, liver; MF, muscular
fibres; N, notochord; OE, oesophagus; OG, oil globule; P, pancreas; PAS, periodic acid-Schiff staining technique; SB, swim bladder; ST, stomach;
YS, yolk sac.
 B.N. Chen et al. / Aquaculture 256 (2006) 489–501 495

Fig. 5. Sagittal section of the stomach of yellowtail kingfish larvae. (a) Larval stomach on 5days after hatching (DAH). Note the gall bladder
(asterisk); HE. Inset picture: detail of the stomach epithelium and the circular muscle layer (bar = 100μm); HE. (b) General view of the stomach on
8 DAH. Note the mucous cells (arrowheads) in the oesophagus; HE. (c) Cardiac and pyloric stomach on 12 DAH. Note the pyloric sphincter
(arrowhead) and mucous cells (arrow); HE. (d) Gastric glands (arrows) in the middle of the stomach on 15 DAH; PAS. Inset picture: gastric glands
(arrowheads, bar = 100 μm); HE. (e) Fundic stomach on 18 DAH. Note gastric glands (arrowheads); HE. (f ) Gastric glands in cardiac and fundic
regions, but not in pyloric region of the stomach on 24 DAH; HE. Inset picture: gastric glands in stomach on 36 DAH (bar = 100μm); HE.
Abbreviations: BC, buccopharyngeal cavity; CM, circular striated muscles; CS, cardiac stomach; FS, fundic stomach; GG, gastric glands; HE,
haematoxylin-eosin; K, kidney; L, liver; MF, muscular fibres; MG, midgut; OE, oesophagus; OG, oil globule; P, pancreas; PAS, periodic acid-Schiff
staining technique; PC, pyloric caeca; PS, pyloric stomach; SB; swim bladder; ST, stomach; Y, yolk residue.

posterior midgut. After the brush border formed on
8 DAH, lipid vacuoles occurred in the enterocytes of the
posterior midgut. Meanwhile, more intestinal folds
developed in both midgut and hindgut, and more
supranuclear vacuoles occurred in the hindgut.
On 12 DAH, numerous lipid vacuoles appeared at the
apical part of the enterocytes in the posterior midgut,
coinciding with fish feeding on Artemia nauplii (Fig.
6d). On the same day, numerous supranuclear vacuoles
occurred in the hindgut, stained in red by the Masson
trichrome stain (Fig. 6d,e). After the formation of a short
rectum in lacking acidophilic supranuclear vacuoles in
enterocytes (Fig. 6e), the number of supranuclear
vacuoles decreased in hindgut, whereas the number of
lipid vacuoles increased in posterior midgut by 18 DAH
(Fig. 6f).
After pyloric caeca buds appeared on the anterior
midgut on 15 DAH (Fig. 7a), rudimentary pyloric caeca
were found on 18 DAH (Fig. 5e). The epithelium on the
pyloric caeca was similar to that on the anterior midgut
(Fig. 7c). Goblet cells were first found in the midgut on
12 DAH (Fig. 7b) and subsequently appeared in hindgut
and pyloric caeca.
3.6. Liver and pancreas
One day after hatching, the liver was a cluster of
spherical shaped cells behind the yolk sac, but liver cells
substantially increased in number by 3 DAH (Fig. 4b).
Blood cells and liver sinusoids were observed on 5 DAH
(Fig. 6c). The gall bladder with a single layer of
columnar cells was found between the liver and the
496 B.N. Chen et al. / Aquaculture 256 (2006) 489–501

Fig. 6. Sagittal section of the digestive tract of yellowtail kingfish larvae. (a) Incipient intestine with a narrow lumen on 0day after hatching (DAH);
HE. (b) Larval fish gut on 4 DAH and the differentiated hindgut; HE. Inset picture: acidophilic supranuclear vacuoles (arrows) in the hindgut
enterocytes (bar = 100μm); HE. (c) Anterior midgut and stomach on 5 DAH. Note the lipid vacuoles (arrows) in the enterocytes; HE. (d) Lipid
vacuoles (arrows) in the posterior midgut and supranuclear vacuoles (arrowheads) in the hindgut on 12 DAH; Masson trichrome. (e) Hindgut on 12
DAH showing the short rectum without folds and supranuclear vacuoles (arrowheads); Masson trichrome. (f) Hindgut on 18 DAH showing the faeces
in the rectum; HE. Inset picture: no supranuclear vacuoles in the enterocytes of hindgut at this stage (bar = 100μm); HE. Abbreviations: AN, anus;
EN, enterocytes; F, faeces; GB, gall bladder; HE, haematoxylin-eosin; HG, hindgut; IN, incipient intestine; IV, intestinal valve; K kidney; L, liver;
MF, muscular fibres; MG, midgut; N, notochord; P, pancreas; R, rectum; SB, swim bladder; SNV, supranuclear vacuoles; ST, stomach; UB, urine
bladder; YS, yolk sac.

pancreas (Figs. 5a and 6c). The hepatocyte cells were
more defined in a spherical shape on 8 DAH (Fig. 5b).
On 12 DAH, the hepatocytes became more contiguous
(Fig. 5c) compared with those in the early stage.
Coinciding with fish feeding on Artemia nauplii on 15
DAH, numerous vacuoles appeared in the liver for
glycogen and lipid storage (Fig. 7d), and the hepatocyte
cytoplasm was full of lipid vacuoles by 36 DAH.
A cluster of basophilic pancreatic cells appeared on 1
DAH adjacent to the incipient intestine behind the liver
and swim bladder. On 3 DAH, the exocrine pancreatic
cells concentrated in acini as pancreatic ducts appeared;
acidophilic zymogen granules were also apparent in the
centre of acini (Fig. 4b). After first feeding, the size of
pancreas, and the number of acini and zymogen
granules in the pancreas increased rapidly on 8 DAH
(Fig. 5b). On 12 DAH, the exocrine pancreas was
located in the gut loop and diffused in the abdominal
cavity along with the fish gut (Fig. 6d). The exocrine
pancreas increased in size as larval fish grew, but no
major change occurred by 36 DAH. The timing of the
major developmental events of the digestive system in
yellowtail kingfish is summarized in Fig. 8.
4. Discussion
4.1. Development of the yellowtail kingfish digestive
system
Similar to other marine fishes (Boulhic and Gabau-
dan, 1992; Bisbal and Bengtson, 1995), the develop-
ment of the digestive system of yellowtail kingfish was
 B.N. Chen et al. / Aquaculture 256 (2006) 489–501 497

Fig. 7. Sagittal section of the pyloric caeca, goblet cells, liver and pancreas. (a) Pyloric caeca bud (arrowhead) at the anterior end of midgut on 15 days
after hatching (DAH); HE. (b) Goblet cells (arrows) in the midgut on 12 DAH. Note supranuclear vacuoles (arrowheads) in the hindgut; PAS. (c)
Goblet cells (arrows) in the pyloric caeca on 36 DAH; PAS. (d) Liver and pancreas on 15 DAH. Note the hepatocytes (arrows) and vacuoles for lipid
storage (arrowheads) in the liver and zymogen granules (asterisks) in the pancreas; HE. Abbreviations: HE, haematoxylin-eosin; HG, hindgut; IV,
intestinal valve; L, liver; MF, muscular fibres; MG, midgut; P, pancreas; PAS, periodic acid-Schiff staining technique; PC, pyloric caeca; ST, stomach.

divided into three phases based on the morphological
and histological characteristics. The first phase started
from hatching and ended on 2 DAH. At the end of this
phase, the mouth opened and the gut was ready to accept
food. The duration of the first phase in yellowtail
kingfish is similar to Dover sole Solea solea L, where
nutrition was solely endogenous in the first 2days
(Boulhic and Gabaudan, 1992), but it was shorter than
common dentex Dentex dentex L., where nutrition was
solely endogenous for 4 days (Santamaria et al., 2004).
The second phase covers the critical time in a larval
life because fish need to develop mechanisms to adapt to
exogenous feeding (May, 1974; Segner et al., 1993).
During this transitional period, yellowtail kingfish larvae

Ontogeny

Pyloric caeca appeared on 18 DAH

Fundic stomach formed on 18 DAH
Gastric glands appeared on 15 DAH
Goblet cells in the intestine appeared on 12 DAH
Gut curve started on 8 DAH
Stomach formed on 5 DAH
SNV appeared on 4 DAH
Intestinal valve formed on 4 DAH
Mouth opened on 2 DAH
Liver and pancreas formed on 1 DAH
Anus opened on 0 DAH
Incipient Intestine existed on 0 DAH

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Days after hatching

Fig. 8. Summary of the major developmental events of the digestive system ontogeny in yellowtail kingfish from hatching to 36days after hatching
(DAH).
498 B.N. Chen et al. / Aquaculture 256 (2006) 489–501
advanced the digestive system in structure and function
for successful ingestion, digestion and assimilation on
exogenous food before the depletion of nutrition re-
serves. As a result, the larval digestive tract was further
differentiated and supranuclear vacuoles and lipid
vacuoles appeared in the hindgut and midgut by 5
DAH after depletion of yolk residuals. Although
important developmental events happened in this
phase, yellowtail kingfish still relied on rotifers and
Artemia nauplii for nutrition. The marked changes in the
digestive system and rapid depletion of yolk and oil
globules may lead larvae to starvation or malnutrition if
suitable food is not available.
The occurrence of gastric glands and pyloric caeca
marked the third developmental phase, which has been
used as a criterion to distinguish larvae from juveniles
(Baglole et al., 1997). To develop from hatching to this
phase, it took 22days in Dover sole (Boulhic and
Gabaudan, 1992), 36 days in yellowtail flounder Pleu-
ronectes ferruginea (Baglole et al., 1997) and 33days in
haddock Melanogrammus aeglefinnus L. (Hamlin et al.,
2000). In contrast, it only took 15 days in yellowtail
kingfish from hatching to develop into the third phase,
indicating the rapid development of its digestive system
over other species.
4.2. Segmentation of the digestive tract
Resembling the development of yellowtail flounder
(Baglole et al., 1997), spotted sand bass Paralabrax
maculatofasciatus (Pena et al., 2003), gilthead sea bream
(Elbal et al., 2004) and common dentex (Santamaria et
al., 2004), the digestive tract of yellowtail kingfish larvae
was an undifferentiated tube before exogenous feeding.
The intestinal valve divided the intestine into midgut and
hindgut by 4 DAH. According to Watanabe (1984), the
appearance of supranuclear vacuoles indicates pinocy-
tosis and intracellular digestion on protein. In this study,
protein digestion might start on 4 DAH as indicated by a
few large acidophilic supranuclear vacuoles in the
hindgut. The formation of presumptive stomach and
pyloric sphincter further divided the digestive tract into
buccopharyngeal cavity, oesophagus, presumptive stom-
ach, midgut and hindgut by 5 DAH. Furthermore, lipid
vacuoles were observed in enterocytes in the anterior
midgut, indicating the possible lipid absorption in the
fish gut (Deplano et al., 1991; Sarasquete et al., 1995;
Pena et al., 2003).
Anatomically, the rectum differentiated from the
hindgut at the end of the digestive tract on 12 DAH. The
histological structure of the rectum in yellowtail
kingfish is similar to that in gilthead sea bream in
lacking of acidophilic supranuclear vacuoles in enter-
ocytes (Calzada et al., 1998), indicating the limited
function in rectum for nutrient absorption. Along with
gut elongation and loop formation, the amount of the
lipid vacuoles in enterocytes reduced in the anterior
midgut and increased in the posterior midgut, which
indicates that the main area for lipid absorption may
have been shifted to the posterior gut.
4.3. Stomach, gastric glands and pyloric caeca
The appearance of gastric glands marked the for-
mation of a functional stomach (Stroband and Kroon,
1981), which is also a histological criterion to
differentiate larvae from juveniles (Tanaka, 1971;
Sarasquete et al., 1995). A functional stomach was
found to breakdown proteins, especially the denatured
protein, to free amino acids by the action of pepsin in an
acid environment and broaden food spectrum of fish
(Segner et al., 1994). For example, weaning onto dry
food can be successfully achieved only after the
stomach has become functional in turbot (Segner et
al., 1993). Gastric glands increase digestive efficiency,
but the timing of gastric gland development varies
greatly among fish species. In slow-growing species, for
instance, gastric glands occurred on 36 DAH in
yellowtail flounder (Baglole et al., 1997) and on 60
DAH in gilthead sea bream (Elbal et al., 2004). In fast-
growing species, on the other hand, gastric glands
occurred on 16 DAH in spotted sand bass (Pena et al.,
2003), and on 11 DAH in Pacific bluefin tuna (Kaji et
al., 1996). Umeda et al. (1987) found that gastric glands
developed after 12 DAH in S. lalandi which is 3 days
earlier than what we found in the present study.
Nevertheless, the quick organ development indicates
the fast-growing potential of yellowtail kingfish.
The formation of the pyloric caeca indicates the last
major change of the digestive system in fish larvae
(Bisbal and Bengtson, 1995; Hamlin et al., 2000). The
pyloric caeca increase the area for digestion and
absorption without increasing the size of intestine and
strengthen the intestinal functions in a limited space of
the abdominal cavity (Buddington and Diamond, 1987;
Bisbal and Bengtson, 1995; Baglole et al., 1997). The
pyloric caeca and the anterior intestine are structurally
similar and have the same function in digestion (Cataldi
et al., 1987). Our histological study showed that the
pyloric caeca developed from the buds in the anterior
midgut and their epitheliums resembled the ones in the
anterior midgut. Furthermore, we observed that the
distribution of goblet cells in both the pyloric caeca and
the anterior midgut was similar. According to
 B.N. Chen et al. / Aquaculture 256 (2006) 489–501
Buddington and Diamond (1987), the function of the
pyloric caeca is triggered by passing particles from the
stomach into the pyloric caeca. This supports the fact that
the pyloric caeca formed on 18 DAH, which was 3days
later than the formation of gastric glands in yellowtail
kingfish.
4.4. Supranuclear vacuoles, goblet cells and weaning
The acidophilic supranuclear vacuoles in the hindgut
signal the beginning of pinocytosis and intracellular
digestion on protein before gastric glands appear
(Watanabe, 1984; Calzada et al., 1998; Hamlin et al.,
2000). Gisbert et al. (2004) reported that the number of
acidophilic supranuclear vacuoles in California halibut
Paralichthys californicus decreased after the gastric
glands were differentiated. In common dentex, the
number and size of supranuclear vacuoles in the hindgut
also decreased progressively after reaching the maxi-
mum on 11 DAH, and disappeared on 18 DAH when
gastric glands formed (Santamaria et al., 2004). In this
study, supranuclear vacuoles appeared on 4 DAH, but
the number decreased after gastric glands formed on 15
DAH. Furthermore, no supranuclear vacuoles were
observed in the hindgut after 18 DAH. This event
indicates that the protein digestion mechanism has
changed from pinocytosis and intracellular digestion to
extracellular digestion when gastric glands start to
produce enzymes.
Goblet cells secrete mucus to facilitate the passage of
food through the oesophagus (Kapoor et al., 1975) and
protect the mucosa against proteolytic degradation by
neutralising stomach acidity (Smith, 1989; Scocco et al.,
1996). In the present study, goblet cells were first
observed on 5 DAH in the oesophagus after 2 days when
larvae could ingest rotifers. The number of goblet cells in
the oesophagus, midgut and hindgut increased consid-
erably by 15 DAH, coinciding with the occurrence of the
gastric glands in the stomach. Because the oesophagus is
so close to the stomach, the mucous secretion from the
goblet cells in the oesophagus may also protect stomach
from proteolytic degradation. Gisbert et al. (2004) found
the goblet cells in the fundic region of the stomach in
California halibut by 30 days, but we did not find goblet
cells in the stomach in yellowtail kingfish by 36 days. In
the present study, we observed that the number of goblet
cells in the pyloric caeca was similar to those in the
midgut in yellowtail kingfish, whereas Hamlin et al.
(2000) reported that goblet cells in the pyloric caeca
were more than those in the intestine in haddock,
indicating that the distribution of goblet cells in the
digestive tract varies among species.
499
The weaning of yellowtail kingfish were started on
18 DAH when the fundic stomach and pyloric caeca
formed. However, some other studies suggest that
weaning should start after the appearance of gastric
glands (Segner et al., 1993; Gordon and Hecht, 2002).
Our data indicate that the formation of the fundic
stomach should be an important indicator for stomach
function because the fundic stomach not only embeds
gastric glands but also provides more space to hold food
for a long period to mix the food well with digestive
enzymes.
In conclusion, the digestive system ontogeny of
yellowtail kingfish larvae follows a similar pattern as for
other marine finfish species. However, the time span of
each developmental phase was much shorter, indicating
the fast growing nature of this fish species. The digestive
system was ready to process exogenous food by the end
of the first developmental phase, but the digestive
system became mature when gastric glands and pyloric
caeca appeared by the onset of the third phase. The
formation of the fundic stomach was suggested to be the
point to wean yellowtail kingfish onto commercial
pellets. Our findings on the development of the digestive
system in yellowtail kingfish should lead to a better
understanding of the ontogeny of fast-growing fish
larvae. Such information is useful to improve the rearing
techniques in marine finfish hatcheries.
Acknowledgements
This research was sponsored by the Playford
Memorial Trust (South Australia) and Aquafin CRC.
We would like to thank Prof. Peter Fairweather for
commenting on the early draft, Ms. Mitchell Lewis for
kindly providing facilities and advice on the histological
study, and Mr. Arron Strawbridge (South Australian
Aquatic Sciences Centre) for skilful assistance in larval
fish rearing.
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