Aquaculture Research, 2017, 48, 4533–4544 doi:10.1111/are.

13278

Ontogenetic development of digestive enzymes in

larval and juvenile crimson snapper Lutjanus
erythopterus (Bloch 1790)

Ke Cui1,2,3, Dachuan Cheng2, Zhenhua Ma2,4, Jian G. Qin4, Shigui Jiang2, Dianrong Sun2 &
Shengwei Ma2
1
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China
2
Tropical Aquaculture Research and Development Center, South China Sea Fisheries Research Institute, Chinese
Academy of Fishery Sciences, Sanya, China
3
College of Animal Science, South China Agricultural University, Guangzhou, China
4
School of Biological Sciences, Flinders University, Adelaide, SA, Australia

Correspondence: Z Ma and D Sun, Tropical Aquaculture Research and Development Center, South China Sea Fisheries Research
Institute, Chinese Academy of Fishery Sciences, Sanya, China. E-mails: zhenhua.ma@hotmail.com; drsun73@163.com

stage and a guide to feeding and weaning of this

Abstract
Ontogenetic development of digestive system in
crimson snapper (Lutjanus erythopterus Bloch
1790) larvae was histologically and enzymatically
(alkaline phosphatase, amylase, lipase, pepsin,
trypsin) examined from hatching to 36 days post
hatching (DPH). The ontogenetic development of
crimson snapper larval fish ontogeny was divided
into three distinct phases: phase I starting from
hatch to the onset of exogenous feeding, phase II
starting from first feeding (2–3 DPH) until the for-
mation of gastric glands (13–14 DPH) and phase
III beginning from the appearance of gastric
glands and continuing onwards. The specific activ-
ities of amylase, lipase, trypsin showed sharp
increase and reached to the maximum from hatch
to 4 DPH, 10 DPH, 20 DPH, respectively, followed
by a declining trend with irregular fluctuation. In
contrast to other enzymes, the specific activities of
alkaline phosphatase showed a gradual increase
from hatch to 29 DPH, followed by a sharp
increase towards 36 DPH. The specific activity of
pepsin was firstly detected on 17 DPH and gradu-
ally increased towards the end of this study. The
total activities of these five enzymes showed a
gradual increase till 29 DPH, followed by a sharp
increase towards 36 DPH except for amylase and
lipase reaching maximum at 32 DPH. The present
study provides better understanding of the diges-
tive ontogeny of crimson snapper during the larval
economically important fish in hatcheries.
Keywords: ontogenetic development, digestive
system, histology, enzyme activity, crimson snap-
per Lutjanus erythopterus
Introduction
In marine fish aquaculture, the early larval stage
is a bottleneck for large-scale production of marine
fish species (Watanabe & Kiron 1994). In larval
fish, even a short period of starvation after the
onset of exogenous feeding can cause nutritional
problems, leading to mortality and deformity in
the early stage (Kjørsvik, van der Meeren, Kryvi,
Arnfinnson & Kvenseth 1991). The digestive phys-
iology during early ontogeny of many fish species
has been studied (Oozeki & Bailey 1995; Perez-
Casanova, Murray & Gallant 2006; Lazo, Men-
doza, Holt, Aguilera & Arnold 2007; Suzer,
Akt€ul€
unb, Cß obana, Kamacıa, Sakaa, Fıratc & Alp-
baza 2007). Some studies have combined histolog-
ical evidence of organ development with
physiological functionality (Lazo, Darias & Gisbert
2011). Functional studies with biochemical and
physiological approach together with structural
development are needed to contribute to the
understanding of digestive capacity of developing
larvae (Kurokawa, Suzuki, Ohta, Kagawa, Tanaka
& Unuma 2002; Darias, Murray, Martinez-

© 2017 John Wiley & Sons Ltd 4533

Digestive functionality in larval crimson snapper K Cui et al.
Rodriguez, Cardenas & Yufera 2005; Srichanun,
Tantikitti, Utarabhand & Kortner 2013). Conse-
quently, structural and functional knowledge of
the digestive system of a fish species during onto-
genetic development is an essential prerequisite for
larval fish rearing in a hatchery (Kjørsvik, Pittman
& Pavlov 2004).
Inappropriate feed supply is one of the factors
leading to massive mortality during the larval
stage (Hunt von Herbing & Gallager 2000;
Rønnestad, Y ufera, Uebersch€ar, Ribeiro, Sæle &
Boglione 2013; Ma, Qin, Hutchinson, Chen &
Song 2014). Larvae with inappropriate nutrition
usually exhibit histological degeneration, especially
in the digestive tract and associated glands (Struss-
mann & Takashima 1989; Y ufera, Pascual, Polo &
Sarasquete 1993), and re-fed larvae cannot digest
food and die even with the food crammed in the
gut (Kjørsvik et al. 1991). Starvation usually
results in shrinkage of enterocytes and reduction
in the height of enterocyte cells in both the midgut
and hindgut (Theilacker & Watanabe 1989; Bisbal
& Bengtson 1995), lack of supranuclear vacuoles
in the hindgut (Crespo, Marin de Mateo, Santa-
maria, Sala, Grau & Pastor 2001), degeneration of
the cellular structure in both liver and pancreas
(Kjørsvik et al. 1991) and malfunction of trunk
musculature (Bisbal & Bengtson 1995; Gisbert,
Conklin & Piedrahita 2004).
Generally, in most teleost fish, ontogenetic
development of the digestive system can be divided
into three major stages (Buddington 1985; Boulhic
& Gabaudan 1992; Bisbal & Bengtson 1995). The
first stage starts from hatch to the completion of
endogenous feeding. In this stage, larval survival
depends on energy reserve in the yolk sac and oil
globules. Larvae experience a transition of endoge-
nous to exogenous feeding before exclusively feed-
ing on external food at the end of the first stage.
The second stage begins with the onset of exoge-
nous feeding and ends before the formation of gas-
tric glands in the stomach, characterized by the
lack of sufficient digestive capabilities (Buddington
1985; Boulhic & Gabaudan 1992). During this
phase, the nutritional requirement of larvae
mainly relies on pinocytosis and intracellular
digestion and absorption (Watanabe 1982; Avila
& Juario 1987; Fukuhara 1988). Usually, fish lar-
vae feed on live food such as rotifers that can be
easily ingested and digested. The third stage starts
from the presence of gastric glands and pyloric
caeca to metamorphosis onwards, indicating the
4534
Aquaculture Research, 2017, 48, 4533–4544
functional maturation of the digestive system
(Tanaka 1971; Bisbal & Bengtson 1995). Coincid-
ing with metamorphosis, the digestive system is
anatomically and physiologically ready to accept
artificial pellets (Bisbal & Bengtson 1995; Gordon
& Hecht 2002). Hence, the understanding of fish
digestive capacity, nutritional requirement and live
food supply will provide useful knowledge to
improve survival and nutrition supplement to fish
larvae (Alvarez-Gonzalez, Cervantes-Trujano,
Tovar-Ramirez, Conklin, Nolasco, Gisbert & Piedra-
hita 2006; Ma, Qin & Nie 2012). In practice, pre-
cise knowledge on the timing of major
developmental events in fish larvae will allow us
to develop an appropriate protocol to wean fish
larvae to compound diets (Baglole, Murray, Goff &
Wright 1997; Cahu & Zambonino-Infante 2001).
Crimson snapper (Lutjanus erythopterus) is one of
the commercially important species for aquacul-
ture and has been successfully reared for decades
in Asia. But high larval mortality (>70%) and
body malformation have hindered industry devel-
opment. Therefore, the study of larval fish diges-
tive physiology should provide information to
solve some of the potential puzzle in the larval fish
culture of this species. Alkaline phosphatase, amy-
lase, lipase and pepsin play important roles in lar-
val fish digestive system during the early
development (reviewed by Lazo et al. 2011). In
this study, we examined the ontogenetic develop-
ment of trypsin, alkaline phosphatase, amylase,
lipase and pepsin and the changes in the histologi-
cal structure in crimson snapper larvae. We aimed
to quantify and evaluate the activity of these
digestive enzymes at each developmental stage
and bridge the relationship between the morpho-
logical change and functional change of the diges-
tive system in crimson snapper.
Materials and methods
Ethics statement
In this study, handling of fish was carried out in
strict accordance with the recommendation in the
Animal Welfare of Chinese Academy of Fishery
Sciences. The protocol, species and number of ani-
mals used in this study were approved by the South
China Sea Fisheries Research Institute Animal Wel-
fare Committee (Approved Number: 2014YJ01).
Fish were euthanized by exposure to an overdose
(40 mg L 1) of AQUI-Sâ (New Zealand).
© 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544
Aquaculture Research, 2017, 48, 4533–4544
Source of fish larvae and experimental conditions
Fertilized eggs of crimson snapper were obtained
from Shenzhen Longqizhuang Aquaculture Hatch-
ery, Guangdong Province, P.R. China, and were
transported to Shenzhen R&D Station of South
China Sea Fisheries Research Institute and
hatched in 500-L fibreglass incubators at 27.5°C
with a hatching rate of 93.5  1.9%
(mean  SD). On 2 days post hatching (DPH), lar-
vae were stocked into three 2500-L larval rearing
tanks at a density of 60 fish L 1. Rearing tanks
were supplied with filtered seawater (5-lm pores)
from the bottom of each tank through upwelling
with a daily exchange rate of 200% tank volume.
Water was discharged through an outlet screen
(300 lm) on the upper side of each tank, and the
screen was cleaned daily to reduce clogging. To
maintain dissolved oxygen close to saturation,
eight air stones were used in each tank. Light
intensity was maintained at 2000 lux, and
the light regime was controlled at 14-h light
and 10-h dark. The salinity was maintained at
33  0.8& and the rearing temperature was
29.0  1.0°C throughout the experiment. Rotifers
Brachionus rotundiformis at a density of 10–
20 rotifers mL 1 were used to feed the larvae
from 2 DPH to 10 DPH. The rotifers fed with
baker yeast were enriched with the S. presso
(INVE Aquaculture, Salt Lake City, UT, USA) for
12 h before the rotifers were added into the lar-
val rearing tanks. Instant microalgal paste (Nan-
nochloropsis sp.; Qingdao Hong Bang Biological
Technology, Qingdao, China) was also added into
larval fish tanks to create a green-water back-
ground.
On 9 DPH, Artemia nauplii were first introduced
at 0.1 nauplii mL 1 and then added with a daily
increment of 90% by number. Starting from 19
DPH, A. nauplii were gradually phased out at a
daily reduction in 20% by number until the co-
feeding period ended. Artemia nauplii were enriched
with DHA Protein Selco (INVE Aquaculture) fol-
lowing the manufacturer’s instruction before add-
ing them to each tank. Three sizes of the inert diet
were used starting from small to large, including
Otohime A1 (~250 lm; Marubeni Nisshin Feed,
Tokyo, Japan), Huacheng No. 3 (360–600 lm)
and Huacheng No. 5 (850–1100 lm; Haikang
Aquatic Biotechnology, Yantai, China). The inert
diets were delivered by hand at a one-hour inter-
val from 08:30 to 19:00 hours daily, and the
© 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544
Digestive functionality in larval crimson snapper K Cui et al.
amount of feed was adjusted to reach the level of
apparent satiation. Each tank bottom was
siphoned daily to remove dead fish, uneaten food
and faeces. The growth stage differentiation and
feeding protocols followed the description in Fig. 1.
Fish sampling and growth measurements
On each sampling day, the sampling time was
scheduled in the morning prior to fish feeding.
Fish larvae were randomly sampled in triplicate
from three different rearing tanks on each day
from hatch to 4 DPH, and then fish specimens
were collected every 2–4 days until the experiment
was finished to examine the development of the
digestive system and digestive enzymes. On each
sampling day, 10–200 fish larvae were randomly
collected from each tank, and fish were anaes-
thetized in 5% AQUI-S (AQUI-S New Zealand,
Lower Hutt, New Zealand) when larvae had been
settled on the bottom of the sampling vials. The
standard length of fish was measured from the
upper jaw to the end of the vertebral column
under a stereo microscope to the nearest
0.05 mm.
Histological analysis procedures
On each of the sampling days, 20 fish larvae from
each rearing tanks were fixed in 10% neutrally
buffered formalin after being anaesthetized in 5%
AQUI-S. Then the fixed fish were individually
embedded in paraffin blocks and sectioned in serial
35
30
Standard length (mm)
25
20 Inert diet
15
Artemia nauplii
10
Rotifer
5 Stage III
Stage I Stage II
0
0 10 20 30 40
DPH
Figure 1 Standard length of crimson snapper from 1
to 36 days post hatch (DPH). Feeding protocol is pre-
sented above the growth curve. Developmental phases
are shown as phases I, II and III, and the diet types are
shown above the growth curve.
4535
Digestive functionality in larval crimson snapper K Cui et al.
sagittal section (5 lm thick) using a Leica RM
2135 rotary microtome (Nussloch, Germany). The
haematoxylin–eosin stain was used for general his-
tological observations. The slide with sections was
mounted permanently using DePex (Shanghai,
China). The sections of five randomly selected fish
were examined under a light microscope. Pho-
tographs were taken with an Olympus digital cam-
era attached to the microscope (Shanghai, China).
Enzymatic assays preparation and procedure
To minimize the potential effect of exogenous
enzymes from undigested prey in the fish gut, on
each sampling day, the sampling time was sched-
uled in the morning prior to fish feeding. After sam-
pling, the fish specimens were thoroughly rinsed in
distilled water to remove external salt and then
immediately stored in liquid nitrogen. The whole
fish was homogenized for enzymatic assays. For
each assay, a pooled sample (5–100 individuals)
of frozen fish was thawed, weighed and homoge-
nized using a glass homogenizer on ice in 0.2 M
NaCl (w/v; Gawlicka, Parent, Horn, Ross, Opstad
& Torrissen 2000). The suspensions were cen-
trifuged at 13 300 g for 10 min at 2°C. Then, the
supernatant was incubated in the enzyme sub-
strate under 25 or 37°C and read on a spec-
trophotometer (SpectraMax M5, Molecular
Devices, LLC., Sunnyvale, CA, USA) at the target
wavelength. All the measurements were carried
out in triplicate.
The alkaline phosphatase (E.C. 3.1.3.1) activity
was assayed using a lipase assay kit (Catalog No.
K722-100; BioVision, Milpitas, CA, USA). The kit
uses p-nitrophenyl phosphate as a phosphatase
substrate which turns yellow (kmax = 405 nm)
when dephosphorylated by alkaline phosphatase.
The a-amylase (E.C. 3.2.1.1) activity was mea-
sured using an amylase activity colorimetric assay
kit (Catalog No. K711-100; BioVision). In the
assay, the substrate of ethylidenepNP-G7 was
cleaved to smaller fragments by a-amylase, and
these fragments produced can be acted upon by a-
glucosidase, which causes the ultimate release of
the chromophore measured at 405 nm. Lipase
(E.C. 3.2.1.1) activity was assayed using a lipase
activity assay kit (Catalog No. K722-100; BioVi-
sion). In this assay, lipase hydrolysed a triglyceride
substrate to form glycerol, which was quantified
enzymatically via monitoring a linked change in
the OxiRed probe absorbance at k = 570 nm.
4536
Aquaculture Research, 2017, 48, 4533–4544
Pepsin (E.C. 3.4.23.1) activity was assayed follow-
ing the method of spectrophotometric stop rate
determination (Zaugg & Wood 1976). One unit
activity produced a DA 280 nm of 0.001 per
min at pH 2.0 and 37°C measured as the tri-
chloroacetic acid-soluble product using haemo-
globin as a substrate. Trypsin (E.C. 3.4.21.4)
activity was measured using a trypsin activity
assay kit (Catalog No. K771-100; BioVision). In
the assay, trypsin was cleaved in the substrate
to generate p-nitroaniline, which was detected
at k = 405 nm.
Enzymatic activities were expressed as the total
activity defined as milli-units per larval fish or
units per larval fish (mU larva 1 or U larva 1)
based on the whole-fish homogenate. The specific
activity was expressed as milli-units per milligram
of protein or units per milligram of protein
(mU mg 1 protein or U mg 1 protein). Soluble
protein of crude enzyme extracts was quantified
using the bicinchoninic acid protein assay kit (Cat-
alog No. BCA1 and B9643; Sigma-Aldrich, St.
Louis, MO, USA).
Statistical analysis
The measurement of each variable was presented
as the mean of pooled fish larvae ranging from 5
to 200 individuals on a sampling day. Mean val-
ues of both total activities and specific activities of
each digestive enzyme between sampling dates
were compared with one-way ANOVA (PASW Statis-
tics 18.0; IBM, Chicago, IL, USA). When a
significant difference was found, Tukey’s test
was performed for multiple range comparisons
(P < 0.05). All the data were tested for normality,
homogeneity and independence before ANOVA.
When the homogeneity of variances was violated,
a nonparametric test (the Welch test) was per-
formed.
Results
Development of the digestive system
Upon hatching, the fish digestive tract was a
straight tube lying dorsally to the yolk sac, and
there was no sign of incipient liver and pancreas
at this stage (Fig. 2a). On 2 DPH, the middle part
of the fish digestive tract inflated gradually. The
mouth of the fish larvae began to open and close
on 3 DPH. On 3 DPH, the incipient liver and
© 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544
Aquaculture Research, 2017, 48, 4533–4544 Digestive functionality in larval crimson snapper K Cui et al.

(a) (b)

(c) (d)

(e) (f)

Figure 2 Morphological observations and sagittal sections of the crimson snapper digestive tract. (a) Profile view of
the 2 days post hatch (DPH) larva. (b) Incipient intestine of 3 DPH larva. (c) Profile view of 4 DPH larva, showing
stomach and liver. (d) Larval stomach on 14 DPH, showing pyloric stomach. (e) Larval hindgut on 17 DPH show-
ing gastric glands (arrow heads). (f) Abdomen on 22 DPH. FP, food particles; IN, incipient intestine; LI, liver; NE,
nephridium; MC, mouth cavity; PA, pancreas; PS, pyloric stomach; SB, swim bladder; ST, stomach; YS, yolk sac.
[Colour figure can be viewed at wileyonlinelibrary.com]

pancreas appeared and became distinctive. Fish
started first feeding at this time and rotifers were
found after dissection. At the end of 3 DPH, the
yolk sac was completely absorbed (Fig. 2b). On 4
DPH, the incipient stomach emerged and the swim
bladder formed (Fig. 2c). Gastric glands were first
observed on 14 DPH and were composed of a sin-
gle type of secretory cells beneath the epithelium
between the cardiac and pyloric regions. With the
inflation of the intestine, rudimentary pyloric
caeca were found at the anterior midgut (Fig. 2d).
Between 16 and 17 DPH, the stomach was divided
into cardiac, fundic and pyloric regions. As larvae
grew, the fundic region was elongated and formed
the largest portion of the stomach.
Trypsin
On 1 DPH, the total activity of trypsin was
detected at 0.01  0.01 U larva 1. From 1 to 29
DPH, the total activity of trypsin increased slightly

© 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544 4537
Digestive functionality in larval crimson snapper K Cui et al. Aquaculture Research, 2017, 48, 4533–4544

(P > 0.05). A significant increase was observed on The activity steadily increased from 1.73  1.72
32 DPH at 45.13  6.79 U larva 1. And then the mU larva 1 on 2 DPH to 177.84  24.33 on 14
total activity of trypsin continuous rose (Fig. 3, DPH and then remained around this level towards 29
P < 0.05). DPH (P > 0.05). The total activity reached to the
The specific activity of trypsin was detected on 1 highest level at 1840.58  404.64 mU larva 1 on
DPH, increased rapidly and peaked at 32 DPH (P < 0.05).
0.19  0.07 U mg 1 protein on 20 DPH (P < 0.05, The specific activity of amylase increased sharply
Fig. 3). Then the specific activity decreased progres- from 0 mU mg 1 protein on 1 DPH to 12.34 
sively to a low level at 5.57  1.08 mU mg 1 pro- 4.87 mU mg 1 protein on 4 DPH after the first feed-
tein on 29 DPH, which was similar to the level in ing (P < 0.05, Fig. 5). Starting from 6 DPH, the
newly hatched larvae (P > 0.05). Subsequently, it activity dropped progressively from 4.39  0.82
continuously increased to the similar peak level as it mU mg 1 protein on 8 DPH to 0.54  0.08
was on 20 DPH (P > 0.05) and on 36 DPH. mU mg 1 protein on 36 DPH (P < 0.05).
Lipase Alkaline phosphatase
The development pattern of the total activity of The total activity of alkaline phosphatase was detected
lipase was similar to that of amylase (Fig. 4). The on 1 DPH at 1.78  0.40 mU larva 1 and remained
total activity was 2.31  0.06 mU larva 1 on 1 low until 26 DPH at 217.86  65.13 mU larva 1
DPH and then remained at a low level until 10 (Fig. 6, P > 0.05). Starting from first weaning, the
DPH (P > 0.05). After 10 DPH, the activity of lipase total activity of alkaline phosphatase increased shar-
increased sharply from 143.33  51.05 mU larva 1 ply from 336.71  87.86 mU larva 1 on 29 DPH to
on 12 DPH to 4028.61  547.65 mU larva 1 on 32 7626.496  1237.739 mU larva 1 on 36 DPH
DPH (P < 0.05). (P < 0.05).
The specific activity of lipase was 0.70  0.01 The specific activity of alkaline phosphatase was
mU mg 1 protein on 1 DPH and increased sharply 0.54  0.12 mU mg 1 protein on 1 DPH and
at 5.76  0.73 mU mg 1 protein on 10 DPH remained low until 26 DPH at 0.71  0.27 mU mg 1
(Fig. 4, P < 0.05), which coincided with fish first protein with low fluctuation (Fig. 6, P > 0.05).
feeding on A. nauplii. After the first peak, the specific Then the specific activity increased sharply from
activity sharply decreased until the end of the experi- 0.71  0.19 mU larva 1 on 29 DPH to 3.42  0.56
ment. On 36 DPH, the specific activity of lipase was mU larva 1 on 36 DPH (P < 0.05).
0.55  0.02 mU mg 1 protein, which was similar
Pepsin
to the level in newly hatched larvae (P > 0.05).
The total activity of pepsin was undetectable at
Amylase
hatch, but was first detected on 17 DPH (Fig. 7).
The total activity of amylase was not detected on Then, the total activity of pepsin increased sharply
1 DPH and remained low until 12 DPH (Fig. 5). with the increase in fish age from 183.33  3.69

400 .30
Specific activity of trypsin (U mg–1 protein)

d'
Total activity of trypsin (U larva–1)

.25

300 d' d'

.20
d' d

.15
200 c',d'
c' c' Figure 3 The total activity of
c'
.10
c' trypsin activity in U larva 1 (black
b' b' b' b' .05 circle) and the specific activity of
100 b'
amylase in U mg 1 protein (clear
a' a'
c circle) from 1 to 32 days post
0.00
b
a aa a a aa a a b b b a hatch (DPH). Mean  SD (n = 3)
0 with the same superscript letter is
0 5 10 15 20 25 30 35 40 not significantly different in multi-
Day post hatching ple comparisons (P < 0.05).

4538 © 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544
Aquaculture Research, 2017, 48, 4533–4544 Digestive functionality in larval crimson snapper K Cui et al.

5000 7

Specific activity of lipase (mU mg–1 protein)

e',f' f

Total activity of lipase (mU larva–1)

6
4000 d',e'
e'
d',e'
c',d',e' d',e' 5
d'
3000 d' c',d'
4
c'
Figure 4 The total activity of b' c'
b',c' 3
2000
lipase activity in mU larva 1 (black b'
circle) and the specific activity of e 2
e
lipase in mU mg 1 protein (clear 1000 d
circle) from 1 to 32 days post a'
c c
c,d 1
a'
hatch (DPH). Mean  SD (n = 3) a b
b
aaaa a a
with the same superscript letter is 0 0
not significantly different in multi- 0 5 10 15 20 25 30 35 40
ple comparisons (P < 0.05). Day post hatching

Specific activity of amylase (mU mg–1 protein)

2500 20
e
Total activity of amylase (mU larva–1)

f' 18

2000 16
14
12
1500 f'
d
10
Figure 5 The total activity of 8
amylase activity in mU larva 1 1000
d' d' 6
(black circle) and the specific activ-
c' 4
ity of amylase in mU mg 1 protein 500 a',b',c'
c b' 2
(clear circle) from 1 to 32 days post b' b' b'
b' b' b' b' b'
a' c
hatch (DPH). Mean  SD (n = 3) a a
b b a,b
0
a aa a a a a
with the same superscript letter is 0
not significantly different in multi- 0 5 10 15 20 25 30 35 40
ple comparisons (P < 0.05). Day post hatching

10 000 5

Specific activity of Alkaline phosphatase

Total activity of Alkaline phosphatase

8000 d 4

(mU mg–1 protein)

c'
(mU larva–1)

Figure 6 The total activity of 6000 3

alkaline phosphatase activity in
mU larva 1 (black circle) and the 4000 2
specific activity of alkaline phos- b' b'
phatase in mU mg 1 protein (clear b' b'
2000 a',b' a',b' a',b' a',b' a',b'
circle) from 1 to 32 days post
a' a',b'
a' c
1
a' a',b' a'
hatch (DPH). Mean  SD (n = 3) b
a a a aa a a,b a,b
aaa a a
with the same superscript letter is 0 0
not significantly different in multi- 0 5 10 15 20 25 30 35 40
ple comparisons (P < 0.05). Day post hatching

mU larva 1 to 107.10  15.79 U larva 1 on 36 2.13  3.69 mU mg 1 protein on 17 DPH. After-

DPH (P < 0.05). wards, the specific activity increased with the increase
The specific activity of pepsin in fish larvae was in fish age and reached the maximum at
undetectable at hatch (Fig. 7) and detected at 48.08  6.96 U mg 1 protein on 36 DPH (P < 0.05).

© 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544 4539
Digestive functionality in larval crimson snapper K Cui et al. Aquaculture Research, 2017, 48, 4533–4544

Specific activity of pepsin (mU mg–1 protein)

1.4e+5 60
Total activity of pepsin (mU larva–1)

1.2e+5 e' 50
d
1.0e+5 40

8.0e+4 30
Figure 7 The total activity of pep-
6.0e+4 d' 20
sin activity in mU larva 1 (black
c'
c' b',c' circle) and the specific activity of
4.0e+4 10
a',b' b'
c
pepsin in mU mg 1 protein (clear
a' a' a' a' a' a' a' a' a'
2.0e+4 0 circle) from 1 to 32 days post
b b hatch (DPH). Mean  SD (n = 3)
a a aa a a a a a a a a
0.0 –10 with the same superscript letter is
0 5 10 15 20 25 30 35 40 not significantly different in multi-
Day post hatching ple comparisons (P < 0.05).

favourable feeding environment, light intensity in

Discussion
this study was maintained at 2000 lux, and the
light regime was controlled at 14-h light and 10-h
Histological structure
dark. Rotifers B. rotundiformis at a density of 10–
The anatomical structures of larval fish for inges- 20 rotifers mL 1 were used to feed the larvae on
tion of exogenous food are complex, including eyes 2 DPH to ensure a high prey encountering rate.
and signal reception organs for prey localization, Ontogenies of digestive organs in many marine
mouth, tail and muscle for capture and digestive teleosts have been studied (Kjørsvik et al. 1991;
tract for digestion and absorption (Yufera & Darias Boulhic & Gabaudan 1992; Bisbal & Bengtson
2007). Most marine fish larvae hatch with a rudi- 1995; Sarasquete et al. 1995; Micale et al. 2006;
mentary digestive system, without mouth and Ma, Zheng, Guo, Zhang, Wang, Jiang, Qin &
unpigmented eyes (Kjørsvik et al. 1991; Boulhic & Zhang 2015). The digestive tract is usually a sim-
Gabaudan 1992; Bisbal & Bengtson 1995; Saras- ple undifferentiated straight tube on the yolk sac
quete, Polo & Y ufera 1995; Micale, Garaffo, Gen- before the mouth opening, with an epithelium cov-
ovese, Spedicato & Muglia 2006). In most marine ered by a monostratified layer of columnar or
fish species, the mouth opening along with the cubical cells. In crimson snapper, the posterior
eyes become pigmented (Kvenseth, Pittman & Hel- part of this tube was bending to open in the cau-
vik 1996; Kawamura, Masuma, Tezuka, Koiso, dal region at mouth opening. The yolk sac was
Jimbo & Namba 2003). The retina consists of sin- completely absorbed on 3 DPH within a few hours
gle cones, and the rod cells appear later in the after mouth opening. On 4 DPH, incipient stomach
development (Helvik, Drivenes, Harboe & Seo formed, and intestinal valve divided the intestine
2001; Shand, Hart, Thomas & Partridge 2002). In into midgut and hindgut.
addition, some rostral free neuromasts and olfac- The appearance of gastric glands marked the
tory ciliate receptor cells are forming during formation of a functional stomach (Stroband &
mouth opening (Yin & Blaxter 1987; Boglione, Kroon 1981), which is also a histological criterion
Giganti, Selmo & Cataudella 2003). It is usually to distinguish larvae from juveniles (Tanaka
considered that marine fish at the early stage are 1971; Sarasquete et al. 1995). A functional stom-
mainly visual feeders and vibratory movement and ach was found to break down protein, the dena-
chemical stimuli are also important for prey detec- tured protein particularly, to free amino acids by
tion and to avoid predation (Y ufera & Darias the action of pepsin in an acid environment (Seg-
2007). In crimson snapper, the eyes became pig- ner, Storch, Reinecke, Kloas & Hanke 1994). In
mented and the mouth began to open on 3 DPH. crimson snapper, rudimentary pyloric caeca were
In intensive farming, although plenty of live food found on the anterior midgut on 17 DPH, and
is available in the tank, fish may still suffer from gastric glands were also observed at this stage,
starvation due to poor vision and mouth gape lim- suggesting a functional digestive system has been
itation (Planas & Cunha 1999). To provide a formed.

4540 © 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544
Aquaculture Research, 2017, 48, 4533–4544
Digestive activity
The activity of pancreatic enzymes (trypsin, lipases
and amylase) has been detected in many marine
fish at first feeding and even before the mouth
opening (Zambonino-Infante & Cahu 1994, 2001;
Oozeki & Bailey 1995; Martınez, Moyano, Fernan-
dez-Diaz & Yufera 1999; Ribeiro, Zambonino-
Infante, Cahu & Dinis 1999; Hoenhe-Reitan,
Kjørsvik & Gjellesvik 2001; Cara, Moyano, Carde-
nas, Fernandez-Diaz & Yufera 2003; Ma, Cahu,
Zambonino-Infante, Yu, Duan, Le Gall & Mai
2005; Alvarez-Gonzalez et al. 2006; Bolasina,
Perez & Yamashita 2006; Chen, Qin, Kumar,
Hutchinson & Clarke 2006a). In Pacific threadfin,
the amylase activity was not quantifiable at hatch,
but its activity could be detected before the first
feeding (Kim, Divakaran, Brown & Ostrowski
2001). In the present study, the specific activity of
trypsin and lipase was detected before the onset of
exogenous feeding, and a sharply increasing trend
was observed from 2 to 10 DPH. The specific
activity of amylase was detected on 2 DPH, with
sharp increase to the maximum specific activity on
4 DPH. In species such as sea bream and yellow-
tail kingfish, the activities of trypsin, amylase and
lipase are significantly stronger than those in the
newly hatched larvae after 2-day development
(Cara et al. 2003; Chen, Qin, Kumar, Hutchinson
& Clarke 2006b), while, in Eurasian perch larvae,
the specific activities of trypsin and amylase were
detected after hatch and increased substantially in
the first few days after exogenous feeding (Cuvier-
Peres & Kestemont 2002). The result from the cur-
rent study together with previous published data
suggests that digestive enzymes of fish larvae are
presented at a low level before the onset exoge-
nous feeding and their activities increase to a high
level during exogenous feeding. In contrast to a
taxonomically similar species golden pompano
(Trachinotus ovatus; Ma et al. 2015), the onset
points of trypsin, amylase and lipase in crimson
snapper are triggered by internal mechanisms,
rather than dietary stimulation. The activities of
cytosolic enzymes secreted by the enterocyte
(amino peptidases, acid and alkaline phosphatases,
esterases) are also present at first feeding (Saras-
quete, Polo & Conzalez de Canales 1993; Baglole,
Goff & Write 1998; Zambonino-Infante & Cahu
2001; Cara et al. 2003). In the present study, the
specific activity of alkaline phosphatases was
detected after hatching, and the same level of
© 2017 John Wiley & Sons Ltd, Aquaculture Research, 48, 4533–4544
Digestive functionality in larval crimson snapper K Cui et al.
activity was maintained from 2 DPH to 29 DPH.
The result from our current study together with
other published data suggests that digestive
enzymes are present at a low level upon hatching
and their activities increase to a high level prior to
the onset of exogenous feeding.
According to Walford and Lam (1993), the pep-
sin activity was detected after the formation of
gastric glands in Asian sea bass larvae, indicating
the close relationship between the structure and
functionality of a stomach (Segner et al. 1994).
Unlike adult fish, most larvae lack a stomach and
cannot carry out gastric proteolytic digestion with
pepsin (Jobling 1995). It has been suggested that
alkaline proteases are responsible for protein diges-
tion until the stomach is fully developed (Ribeiro,
Zambonino-Infante, Cahu & Dinos 2002). The
fluctuations in specific enzyme activities covered the
period of morphological differentiation in the diges-
tive tract and the development of digestive glands
(Ma et al. 2015). According to this study, gastric
glands were first observed on 14 DPH, but the pep-
sin activity of crimson snapper was not detected
until 17 DPH. Such development may cause by the
feeds shifting from rotifers to A. nauplii.
To improve larval rearing and feeding practices,
the functional development of digestive system in
several species from the genus of Lutjanus has been
evaluated (Galaviz, Garcia-Ortega, Gisbert, Lopez &
Gasca 2012; Moguel-Hern andez, Pe~ na, Nolasco-
Soria, Duma & Zavala-Leal 2014; Pe~ na, Dumas &
Contreras-Olguin 2016). In Pacific red snapper
(Lutjanus peru), the appearance of the gastric
glands and pyloric caeca was around 24 DPH
(Pe~na et al. 2016), and pepsin secretion was
detected around 25 DPH (Moguel-Hern andez et al.
2014). Based on the development of the digestive
system and enzymes, these authors suggest that
weaning of L. peru should be conducted after 20
DPH. In spotted rose snapper (Lutjanus guttatus),
gastric gland was firstly observed on 20 DPH, and
pepsin was detected in the same day (Galaviz et al.
2012). In the present study, gastric glands in the
stomach of L. erythopterus were observed around
14 DPH, and pepsin was detected after 17 DPH.
The difference in the time of detection of pepsin
activity and gastric glands may be due to the spe-
cies variation. Recent study has demonstrated a
daily rhythms of digestive enzyme activity in fish
larvae (Mata-Sptres, Moyano, Martinez-Rodriguez
& Yufera 2016).The present study was not affected
by this circumstance, as all the sampling time in
4541
Digestive functionality in larval crimson snapper K Cui et al.
this study was scheduled in the morning prior to
fish feeding.
Conclusions
The levels of enzyme activities determined at first
feeding are close to those measured a few days
later in the actively feeding larvae. Although the
amplitude of fluctuation in specific enzyme activi-
ties is large, the total activities of these enzymes
showed a trend of gradual increase with fish age.
The secretion of pepsin on 17 DPH suggests the
readiness of digestive system and the timing for
inert diet introduction. Crimson snapper belongs to
the fast-growing species as the duration of diges-
tive ontogeny of crimson snapper larvae is signifi-
cantly shorter than other slow-growing species.
Our findings on the development of the digestive
system in crimson snapper provide a better under-
standing of the ontogeny of fish larvae, and such
information is also useful to improve the larval
rearing techniques in hatchery practices.
Acknowledgments
This project was funded by China-ASEAN Mar-
itime Cooperation Fund (China and Vietnam Beibu
Gulf Fishery Resources Restocking and Conserva-
tion), Ministry of Agriculture Special Financial
Funding (South China Sea Fisheries Centre) and
Special Scientific Research Funds for Central Non-
profit Institutes, South China Sea Fisheries
Research Institute, Chinese Academy of Fishery
Sciences (2013YD07, 2014YJ01).
Conflicts of interest
The authors declare no conflicts of interest.
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