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Aquaculture 256 (2006) 489 – 501


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
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia
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


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,

ingest, digest and absorb food (Kjorsvik et al., 2004).
Successful development of the digestive system is Although larval fish may be morphologically capable of
crucial for the survival and growth in fish larvae because 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
⁎ Corresponding author. Tel.: +61 8 8201 3045; fax: +61 8 8201
functional (Govoni et al., 1986; Canino and Bailey,
E-mail address: jian.qin@flinders.edu.au (J.G. Qin). 1995). Knowledge on the structural development of the
Present address: South Australian Research and Development digestive system is essential to understand the digestive
Institute, PO Box 120, Henley Beach SA 5022, Australia. physiology and determine the appropriate timing to
0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
490 B.N. Chen et al. / Aquaculture 256 (2006) 489–501

wean fish larvae (Watanabe and Kiron, 1994; Baglole et Dicentrarchus labrax L. (Cahu and Zamboninon
al., 1997; Cahu and Zambonino Infante, 2001). Infante, 1995; Peres et al., 1998).
Ontogenetic development of the digestive system in Yellowtail kingfish has been used for commercial
most teleost fish is generally divided into three major aquaculture in South Australia for its fast growth, high
phases (Buddington, 1985; Boulhic and Gabaudan, flesh quality and suitability for cage culture (Fowler et al.,
1992; Bisbal and Bengtson, 1995). The first phase starts 2003). Although the life cycle is closed in yellowtail
from hatching and ends at the completion of endogenous kingfish (Tachihara et al., 1997; Gillanders et al., 1999;
feeding. During this period, larvae depend on energy Poortenaar et al., 2001), high larval deformities and
reserves in the yolk sac and oil globules. Toward the end mortalities have hindered the industry development.
of the first phase, fish experience a transition of Therefore, the study of larval fish nutritional physiology
endogenous to exogenous feeding before exclusively in this species should provide information to solve some
feeding on external food. The second phase starts from of these problems (Govoni et al., 1986; Segner et al.,
the onset of exogenous feeding and ends before the 1993). However, the study of histological development of
formation of gastric glands in the stomach, characterized the larval fish digestive system should be undertaken as a
by the lack of sufficient digestive capabilities (Budding- first step because most physiological functions are based
ton, 1985; Boulhic and Gabaudan, 1992). During this on the organ development (Hamlin et al., 2000; Govoni,
phase, larval fish mainly depend on pinocytosis and 2004). The objective of this study was therefore to
intracellular digestion and absorption (Watanabe, 1982). examine the structural changes during the ontogeny of the
As a result, fish larvae usually feed on live food such as digestive system of hatchery-reared yellowtail kingfish
rotifers that can be easily ingested and digested. The third from hatching until metamorphosis, aiming to under-
phase starts from the presence of gastric glands and stand the sequence of organ development and provide
pyloric caeca to metamorphosis onward, indicating the fundamental knowledge on hatchery management for
functional maturation of the digestive system (Tanaka, fast-growing species such as yellowtail kingfish.
1971; Bisbal and Bengtson, 1995). The third develop-
mental phase coincides with metamorphosis, when the 2. Materials and methods
digestive system anatomically and physiologically is
ready to accept artificial pellets (Bisbal and Bengtson, 2.1. Eggs and larval fish rearing
1995; Gordon and Hecht, 2002). Despite the overall
similarity of the developmental pattern, the duration of Fertilized eggs of the same batch of brood yellowtail
each developmental phase varies among fish species. kingfish were obtained from the Arno Bay, South
The understanding of ontogenetic development of the Australia, and transported to the South Australian
digestive system is crucial for larval fish rearing in any Aquatic Sciences Centre, Adelaide. Upon arrival, all
economically important aquaculture species. eggs were hatched in 120-L fibreglass incubators at
Fragmental information exists on the histological 21°C. After hatching, the larvae were stocked in 600-L
development of the digestive system of yellowtail fibreglass rearing tanks at 60 fish/L. All rearing tanks
kingfish Seriola lalandi (Umeda et al., 1987) and were supplied with filtered seawater with a 5-μm filter in
another yellowtail species Seriola quinqueradiata in the a flow-through system with a water exchange rate of
same genus (Umeda and Ochiai, 1973, 1975). Umeda et 1.2–3.0 L/min. Two air stones were used in each tank to
al. (1987) found that the liver became functional before maintain dissolved oxygen at saturation and also to
the onset of exogenous feeding in yellowtail kingfish. promote a homogeneous distribution of microalgae and
These authors further noticed that digestion and live foods (rotifers and Artemia nauplii). Light intensity
absorption occurred 3–4 days after the onset of at 3000–6000lx and a photoperiod of 13h light and 11 h
exogenous feeding as evidenced by the lipid droplets dark were provided. Salinity was maintained at 38‰
and supranuclear vacuoles in the intestine enterocytes. throughout the experiment and water temperature was
However, the information about its ontogenetic devel- controlled at 24 ± 1.3°C.
opment of the digestive system is incomplete comparing Rotifers (Brachionus plicatilis) were given to fish
to some other important commercial species such as larvae on 2days after hatching (DAH) until 12 DAH at
turbot Scophthalmus maximus L. (Cousin and Laur- 10 individuals/mL. The rotifers fed with microalgae
encin, 1987; Hoehne-Reitan et al., 2003), gilthead (Nannochloropsis oculata) were enriched with DHA
seabream Sparus aurata L. (Sarasquete et al., 1995; Selco (INVE Aquaculture) for 12 h before being added
Calzada et al., 1998), Senegal sole Solea senegalensis into the fish tanks. A microalga (Isochrysis sp.) was also
(Ribeiro et al., 1999; Arellano et al., 2001) and sea bass added to the larval fish tanks as food for the rotifers.
B.N. Chen et al. / Aquaculture 256 (2006) 489–501 491

Artemia nauplii enriched with dried algae (Bio-Marine Bancroft and Gamble, 2002). The slide with sections
Algamac 3050™) were introduced to the rearing tanks was mounted permanently using DePex. The sections of
from 10 to 24 DAH at 5 individuals/mL. Commercial five fish were randomly examined under an Olympus
pellet feed (Proton 3, INVE Aquaculture) was intro- light microscope. Photographs were taken with a Canon
duced to the fish from 18 DAH to 36 DAH to the end of digital photomicrographic attachment.
the experiment (36 DAH).
3. Results
2.2. Fish sampling and growth measurements
3.1. Fish growth and general development of the
The fish larvae were randomly collected in triplicate digestive system
from the rearing tanks each day from hatching to 5 DAH,
and then specimens were collected on 8, 12, 15, 18, 24 Standard length of larval fish averaged 4.30 mm at
and 36 DAH to examine the development of the hatching (0 DAH), and increased to 22.56 mm on 36
digestive system. Standard length was measured, as a DAH (Fig. 1). The absolute growth rate and specific
mean of 10 fish, from the upper jaw to the end of the growth rate under laboratory conditions were 0.51 mm/
notochord for larvae, or to the end of the vertebral day and 4.60%/day, respectively. Upon hatching, the fish
column for juveniles, under a microscope to the nearest digestive tract was a straight tube lying dorsally to the
0.05 mm on each sampling day. The larval growth was yolk sac with posterior portion bended ventrally (Fig.
determined by the measurement of the absolute growth 2a). The lumen was observed in the posterior of the tube
rate (AGR) as mm/day and specific growth rate (SGR) as and joined the urine bladder at the posterior intestine
%/day (Hopkins, 1992). AGR was calculated as: AGR = (Fig. 2b). This connection was further confirmed by the
(SLf − SLi) / Δt, and SGR was determined as: SGR = 100 observation of liquid discharge from the urine bladder
(LnSLf − LnSLi) / Δt, where SLf and SLi are the final and into the hindgut cavity. Although the anus was open, the
initial fish standard length (mm), respectively, and Δt is mouth was closed on 0 DAH (Fig. 2a,b). There was no
the time interval (days) between samples. sign of incipient liver and pancreas at this stage. Rotifers
were supplied to the fish on 2 DAH. However, we did
2.3. Morphological observation not find any rotifer in the fish gut on that day. Rotifers
were first detected in the fish gut on 3 DAH through the
Fifteen fish larvae from each of three replicate tanks gut analysis. On 4 DAH, food particles were easily
were collected on each sampling day and 5 fish was identified in the midgut (Fig. 2d). Since most oil
randomly examined for the morphology of the digestive globules in the yolk sac depleted by 8 DAH, the mixed
tract under a Leica dissecting microscope and a Nikon feeding should have occurred between 3 and 8 DAH.
microscope. From 0 to 12 DAH, since fish were transparent, Although the growth of larvae was not significant during
all larvae were directly observed under the microscope after 4–8 DAH, mixed feeding should be critical for the
being anaesthetized in Benzocaine solution. However, from development of fish organ during this period.
15 DAH onward, since the body wall became opaque, the After mouth opening on 2 DAH, the digestive tract
digestive tract was dissected out for observation. Photo- elongated and the gut lumen increased in size (Fig. 2c).
graphs were taken with a Canon digital photomicrographic The formation of the intestinal valve on 4 DAH divided
attachment during the observation. the incipient intestine into midgut and hindgut (Fig. 2d).
At this stage, the amount of yolk sac and oil globules
2.4. Histological analysis procedures were reduced, but remained in the anterior abdominal
cavity. The liver was behind the yolk sac. When the gut
On each sampling day, 30 larvae from each of three curve formed on 8 DAH at the anterior midgut, the size
replicate tanks were fixed in Zamboni's fixative after of digestive tract substantially increased (Fig. 2e). The
being anaesthetized in Benzocaine solution. The fixed observation of pyloric caeca on 24 DAH marked the last
fish were individually embedded in paraffin blocks and major morphological change in the digestive tract
sectioned in serial sagittal sections (5-μm thick) using a development (Fig. 2f).
Leica RM 2135 rotary microtome. The haematoxylin-
eosin (HE) stain was used for general histological 3.2. Buccopharynx
observations. Masson trichrome was used to stain
collagen, while the periodic acid-Schiff technique Buccopharyngeal cavity was not formed on 0 DAH
(PAS) was used to stain neutral mucins (Pearse, 1985; (Fig. 3a), but oral valves seen as dorsal and ventral
492 B.N. Chen et al. / Aquaculture 256 (2006) 489–501


Phase Phase Phase


Standard length (mm)


10 Rotifers and
Artemia nauplii
nauplii Compound diet
5 and
Artemia compound
Yolk Rotifers nauplii diet
012 4 5 8 12 15 18 24 36
Days after hatching

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). At first feeding on 3 DAH, no mucous cells and folds
The buccopharyngeal cavity lined by a single layer of were observed in the oesophagus. However, on 5 DAH,
squamous epithelium was observed on 1 DAH before mucous cells were formed in the oesophagus mucosae
mouth opening on 2 DAH. On 4 DAH, a stratified and rapidly increased in number, coinciding with fish
squamous epithelium with connective tissues formed feeding on rotifers at this stage. The mucous cells were
the buccopharyngeal mucosa (Fig. 3c). stained in red by PAS on 12 DAH, indicating the
Pharyngeal tooth occurred on 8 DAH in the presence of neutral glycoproteins or mucosubstances.
pharynx (Fig. 3d), and numerically increased as fish Numerous mucous cells were found in the posterior
grew. Taste buds also appeared in the posterior of the oesophagus on 15 DAH when food was changed from
buccopharyngeal cavity on 8 DAH (Fig. 3d) and rotifers to Artemia nauplii (Fig. 4c, d).
became more numerous on 15 DAH (Fig. 4c). The Two days after exogenous feeding on 5 DAH,
tongue formed on 8 DAH by a simple thickening of longitudinal folds formed along the oesophagus, and
the oral floor consisting of a stratified squamous became abundant on 8 DAH (Fig. 5b). Oesophagus
epithelium (Fig. 3d). mucosal folds formed by a simple cubic epithelium.
More longitudinal folds and mucous cells formed
3.3. Oesophagus between the oesophagus and stomach on 15 DAH
(Fig. 4d), but there were no substantial histological
Upon hatching, the oesophagus appeared as a simple changes from 15 to 36 DAH.
tube similar to the incipient gut, and was lined by a
single layer of simple cuboidal epithelium (Fig. 4a). 3.4. Stomach
Before the formation of the buccopharyngeal cavity, the
digestive tract was closed at the oesophagus. On 3 DAH, The presumptive stomach appeared as a bulge at
the oesophageal lumen lined with a stratified squamous the end of the oesophagus on 4 DAH (Fig. 3c). On 5
epithelium was formed to connect the buccopharyngeal DAH, a preliminary pyloric sphincter developed and
cavity and intestine (Fig. 4b). On 4 DAH, the posterior the mucosae of stomach formed by a simple cubic
oesophagus expanded to form the stomach (Fig. 3c). epithelium similar to that in oesophageal mucosae
The oesophagus was surrounded by a circular layer of (Fig. 5a). On 8 DAH, the stomach became more
striated muscles, and the muscle layer became thicker as elongated and the border between the stomach and
larvae grew. 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 36 DAH no goblet cells were observed in the epithelium
epithelium (Fig. 5b). On 12 DAH, the pyloric sphincter in the stomach.
separated the stomach from the midgut and the
stomach then comprising the cardiac and pyloric re- 3.5. Intestine and pyloric caeca
gions (Fig. 5c).
On 15 DAH, the presence of gastric glands advanced The incipient intestine of a newly-hatched larva was
the presumptive stomach (Fig. 5d), coinciding with fish lined by a single layer of columnar cells (Fig. 6a).
feeding on Artemia nauplii. The gastric glands were Structural changes of the larval intestine occurred
simple acinus glands beneath the epithelium between between 3 and 4 DAH when fish shifted from
the cardiac and pyloric regions (Fig. 5d), and the gastric endogenous nutrition to a mixture of endogenous and
glands were composed of single-type secretory cells and exogenous nutrition. On 4 DAH, the intestinal valve
PAS-positive apical borders. On 18 DAH, the stomach divided the intestine into midgut and hindgut (Fig. 6b).
was further divided into cardiac, fundic and pyloric Subsequently, numerous acidophilic supranuclear
regions (Fig. 5e). The gastric glands scattered in the vacuoles appeared in the hindgut, indicating the start
cardiac and fundic regions but not in the pyloric region of protein digestion and absorption in the gut (Fig. 6b).
(Fig. 5f). The fundic region was elongated as larvae On 5 DAH, lipid vacuoles appeared in the enter-
grew and formed the largest portion of the stomach. By 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 After pyloric caeca buds appeared on the anterior
8 DAH, lipid vacuoles occurred in the enterocytes of the midgut on 15 DAH (Fig. 7a), rudimentary pyloric caeca
posterior midgut. Meanwhile, more intestinal folds were found on 18 DAH (Fig. 5e). The epithelium on the
developed in both midgut and hindgut, and more pyloric caeca was similar to that on the anterior midgut
supranuclear vacuoles occurred in the hindgut. (Fig. 7c). Goblet cells were first found in the midgut on
On 12 DAH, numerous lipid vacuoles appeared at the 12 DAH (Fig. 7b) and subsequently appeared in hindgut
apical part of the enterocytes in the posterior midgut, and pyloric caeca.
coinciding with fish feeding on Artemia nauplii (Fig.
6d). On the same day, numerous supranuclear vacuoles 3.6. Liver and pancreas
occurred in the hindgut, stained in red by the Masson
trichrome stain (Fig. 6d,e). After the formation of a short One day after hatching, the liver was a cluster of
rectum in lacking acidophilic supranuclear vacuoles in spherical shaped cells behind the yolk sac, but liver cells
enterocytes (Fig. 6e), the number of supranuclear substantially increased in number by 3 DAH (Fig. 4b).
vacuoles decreased in hindgut, whereas the number of Blood cells and liver sinusoids were observed on 5 DAH
lipid vacuoles increased in posterior midgut by 18 DAH (Fig. 6c). The gall bladder with a single layer of
(Fig. 6f). 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 (Fig. 5b). On 12 DAH, the exocrine pancreas was
more defined in a spherical shape on 8 DAH (Fig. 5b). located in the gut loop and diffused in the abdominal
On 12 DAH, the hepatocytes became more contiguous cavity along with the fish gut (Fig. 6d). The exocrine
(Fig. 5c) compared with those in the early stage. pancreas increased in size as larval fish grew, but no
Coinciding with fish feeding on Artemia nauplii on 15 major change occurred by 36 DAH. The timing of the
DAH, numerous vacuoles appeared in the liver for major developmental events of the digestive system in
glycogen and lipid storage (Fig. 7d), and the hepatocyte yellowtail kingfish is summarized in Fig. 8.
cytoplasm was full of lipid vacuoles by 36 DAH.
A cluster of basophilic pancreatic cells appeared on 1 4. Discussion
DAH adjacent to the incipient intestine behind the liver
and swim bladder. On 3 DAH, the exocrine pancreatic 4.1. Development of the yellowtail kingfish digestive
cells concentrated in acini as pancreatic ducts appeared; system
acidophilic zymogen granules were also apparent in the
centre of acini (Fig. 4b). After first feeding, the size of Similar to other marine fishes (Boulhic and Gabau-
pancreas, and the number of acini and zymogen dan, 1992; Bisbal and Bengtson, 1995), the develop-
granules in the pancreas increased rapidly on 8 DAH 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 (Boulhic and Gabaudan, 1992), but it was shorter than
and histological characteristics. The first phase started common dentex Dentex dentex L., where nutrition was
from hatching and ended on 2 DAH. At the end of this solely endogenous for 4 days (Santamaria et al., 2004).
phase, the mouth opened and the gut was ready to accept The second phase covers the critical time in a larval
food. The duration of the first phase in yellowtail life because fish need to develop mechanisms to adapt to
kingfish is similar to Dover sole Solea solea L, where exogenous feeding (May, 1974; Segner et al., 1993).
nutrition was solely endogenous in the first 2days During this transitional period, yellowtail kingfish larvae


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
498 B.N. Chen et al. / Aquaculture 256 (2006) 489–501

advanced the digestive system in structure and function lacking of acidophilic supranuclear vacuoles in enter-
for successful ingestion, digestion and assimilation on ocytes (Calzada et al., 1998), indicating the limited
exogenous food before the depletion of nutrition re- function in rectum for nutrient absorption. Along with
serves. As a result, the larval digestive tract was further gut elongation and loop formation, the amount of the
differentiated and supranuclear vacuoles and lipid lipid vacuoles in enterocytes reduced in the anterior
vacuoles appeared in the hindgut and midgut by 5 midgut and increased in the posterior midgut, which
DAH after depletion of yolk residuals. Although indicates that the main area for lipid absorption may
important developmental events happened in this have been shifted to the posterior gut.
phase, yellowtail kingfish still relied on rotifers and
Artemia nauplii for nutrition. The marked changes in the 4.3. Stomach, gastric glands and pyloric caeca
digestive system and rapid depletion of yolk and oil
globules may lead larvae to starvation or malnutrition if The appearance of gastric glands marked the for-
suitable food is not available. mation of a functional stomach (Stroband and Kroon,
The occurrence of gastric glands and pyloric caeca 1981), which is also a histological criterion to
marked the third developmental phase, which has been differentiate larvae from juveniles (Tanaka, 1971;
used as a criterion to distinguish larvae from juveniles Sarasquete et al., 1995). A functional stomach was
(Baglole et al., 1997). To develop from hatching to this found to breakdown proteins, especially the denatured
phase, it took 22days in Dover sole (Boulhic and protein, to free amino acids by the action of pepsin in an
Gabaudan, 1992), 36 days in yellowtail flounder Pleu- acid environment and broaden food spectrum of fish
ronectes ferruginea (Baglole et al., 1997) and 33days in (Segner et al., 1994). For example, weaning onto dry
haddock Melanogrammus aeglefinnus L. (Hamlin et al., food can be successfully achieved only after the
2000). In contrast, it only took 15 days in yellowtail stomach has become functional in turbot (Segner et
kingfish from hatching to develop into the third phase, al., 1993). Gastric glands increase digestive efficiency,
indicating the rapid development of its digestive system but the timing of gastric gland development varies
over other species. greatly among fish species. In slow-growing species, for
instance, gastric glands occurred on 36 DAH in
4.2. Segmentation of the digestive tract yellowtail flounder (Baglole et al., 1997) and on 60
DAH in gilthead sea bream (Elbal et al., 2004). In fast-
Resembling the development of yellowtail flounder growing species, on the other hand, gastric glands
(Baglole et al., 1997), spotted sand bass Paralabrax occurred on 16 DAH in spotted sand bass (Pena et al.,
maculatofasciatus (Pena et al., 2003), gilthead sea bream 2003), and on 11 DAH in Pacific bluefin tuna (Kaji et
(Elbal et al., 2004) and common dentex (Santamaria et al., 1996). Umeda et al. (1987) found that gastric glands
al., 2004), the digestive tract of yellowtail kingfish larvae developed after 12 DAH in S. lalandi which is 3 days
was an undifferentiated tube before exogenous feeding. earlier than what we found in the present study.
The intestinal valve divided the intestine into midgut and Nevertheless, the quick organ development indicates
hindgut by 4 DAH. According to Watanabe (1984), the the fast-growing potential of yellowtail kingfish.
appearance of supranuclear vacuoles indicates pinocy- The formation of the pyloric caeca indicates the last
tosis and intracellular digestion on protein. In this study, major change of the digestive system in fish larvae
protein digestion might start on 4 DAH as indicated by a (Bisbal and Bengtson, 1995; Hamlin et al., 2000). The
few large acidophilic supranuclear vacuoles in the pyloric caeca increase the area for digestion and
hindgut. The formation of presumptive stomach and absorption without increasing the size of intestine and
pyloric sphincter further divided the digestive tract into strengthen the intestinal functions in a limited space of
buccopharyngeal cavity, oesophagus, presumptive stom- the abdominal cavity (Buddington and Diamond, 1987;
ach, midgut and hindgut by 5 DAH. Furthermore, lipid Bisbal and Bengtson, 1995; Baglole et al., 1997). The
vacuoles were observed in enterocytes in the anterior pyloric caeca and the anterior intestine are structurally
midgut, indicating the possible lipid absorption in the similar and have the same function in digestion (Cataldi
fish gut (Deplano et al., 1991; Sarasquete et al., 1995; et al., 1987). Our histological study showed that the
Pena et al., 2003). pyloric caeca developed from the buds in the anterior
Anatomically, the rectum differentiated from the midgut and their epitheliums resembled the ones in the
hindgut at the end of the digestive tract on 12 DAH. The anterior midgut. Furthermore, we observed that the
histological structure of the rectum in yellowtail distribution of goblet cells in both the pyloric caeca and
kingfish is similar to that in gilthead sea bream in the anterior midgut was similar. According to
B.N. Chen et al. / Aquaculture 256 (2006) 489–501 499

Buddington and Diamond (1987), the function of the The weaning of yellowtail kingfish were started on
pyloric caeca is triggered by passing particles from the 18 DAH when the fundic stomach and pyloric caeca
stomach into the pyloric caeca. This supports the fact that formed. However, some other studies suggest that
the pyloric caeca formed on 18 DAH, which was 3days weaning should start after the appearance of gastric
later than the formation of gastric glands in yellowtail glands (Segner et al., 1993; Gordon and Hecht, 2002).
kingfish. Our data indicate that the formation of the fundic
stomach should be an important indicator for stomach
4.4. Supranuclear vacuoles, goblet cells and weaning function because the fundic stomach not only embeds
gastric glands but also provides more space to hold food
The acidophilic supranuclear vacuoles in the hindgut for a long period to mix the food well with digestive
signal the beginning of pinocytosis and intracellular enzymes.
digestion on protein before gastric glands appear In conclusion, the digestive system ontogeny of
(Watanabe, 1984; Calzada et al., 1998; Hamlin et al., yellowtail kingfish larvae follows a similar pattern as for
2000). Gisbert et al. (2004) reported that the number of other marine finfish species. However, the time span of
acidophilic supranuclear vacuoles in California halibut each developmental phase was much shorter, indicating
Paralichthys californicus decreased after the gastric the fast growing nature of this fish species. The digestive
glands were differentiated. In common dentex, the system was ready to process exogenous food by the end
number and size of supranuclear vacuoles in the hindgut of the first developmental phase, but the digestive
also decreased progressively after reaching the maxi- system became mature when gastric glands and pyloric
mum on 11 DAH, and disappeared on 18 DAH when caeca appeared by the onset of the third phase. The
gastric glands formed (Santamaria et al., 2004). In this formation of the fundic stomach was suggested to be the
study, supranuclear vacuoles appeared on 4 DAH, but point to wean yellowtail kingfish onto commercial
the number decreased after gastric glands formed on 15 pellets. Our findings on the development of the digestive
DAH. Furthermore, no supranuclear vacuoles were system in yellowtail kingfish should lead to a better
observed in the hindgut after 18 DAH. This event understanding of the ontogeny of fast-growing fish
indicates that the protein digestion mechanism has larvae. Such information is useful to improve the rearing
changed from pinocytosis and intracellular digestion to techniques in marine finfish hatcheries.
extracellular digestion when gastric glands start to
produce enzymes. Acknowledgements
Goblet cells secrete mucus to facilitate the passage of
food through the oesophagus (Kapoor et al., 1975) and This research was sponsored by the Playford
protect the mucosa against proteolytic degradation by Memorial Trust (South Australia) and Aquafin CRC.
neutralising stomach acidity (Smith, 1989; Scocco et al., We would like to thank Prof. Peter Fairweather for
1996). In the present study, goblet cells were first commenting on the early draft, Ms. Mitchell Lewis for
observed on 5 DAH in the oesophagus after 2 days when kindly providing facilities and advice on the histological
larvae could ingest rotifers. The number of goblet cells in study, and Mr. Arron Strawbridge (South Australian
the oesophagus, midgut and hindgut increased consid- Aquatic Sciences Centre) for skilful assistance in larval
erably by 15 DAH, coinciding with the occurrence of the fish rearing.
gastric glands in the stomach. Because the oesophagus is
so close to the stomach, the mucous secretion from the References
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