Aquaculture Research, 2002, 33, 663±672
Characterization of protease activity in developing
discus Symphysodon aequifasciata larva
Alexander Chong, Roshada Hashim, Leng-Choy Lee & Ahyaudin bin Ali
School of Biological Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia
Correspondence: Alexander Chong, School of Biological Sciences, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia.
E-mail: alex@usm.my
Abstract
The activity of different protease classes was moni-
tored in developing discus (Symphysodon spp.)
larvae using a combination of biochemical assays
and substrate SDS±PAGE techniques. Results
showed the presence of alkaline proteases of serine
proteases such as trypsin with a significant increase
in activity levels detected beginning 3 days after
hatching. Other alkaline proteases such as metallo-
proteases and chymotrypsin, a type of serine prote-
ase, were only detected in older larval stages, at
around 20±30 days after hatching. Acidic protease
activity was very low during the first 20±25 days
of development before showing a significant
(P , 0.01) rise. This is despite the formation of a
stomach observed 10 days after hatching. Based on
the development of the protein digestive system ob-
served, the use of microdiets to replace Artemia
should be considered 25 days after hatching.
Keywords: discus, larva, protease, development,
characterization
Introduction
The process of raising fish larva to the juvenile stage
has been identified as a major bottleneck in attempts
to increase production of numerous fish species
(Alves, Specker & Bengston 1999; Turano, Davis &
Arnold 2000). Most of the problems are associated
with nutrition (Planas & Cunha 1999). The use of
both live feed and dry microdiets faces several prob-
lems needing to be addressed. Artemia and rotifer
ß 2002 Blackwell Science Ltd
usage, for instance, are often hampered by high
cost, irregular supply and the need for infrastructure
and labour to enable mass production (Lavens &
Sorgeloos 2000). Although dry micro particulate
feed has been proposed in numerous reports as a
possible replacement, limited success has been
reported in selected species (Charlon & Bergot
1984; Person-Le Ruyet, Alexandre, Thebauld &
Mugnier 1993; Cahu, Zambonino-Infante, Escaffre,
Bergot & Kaushik 1998; Yufera, Pascual &
Fernandez-Diaz 1999). In most cases, early weaning
of larvae to dry diets resulted in higher mortality
and decreased growth as compared with continuos
feeding with live feed (Verreth, Torreele, Storch &
Segner 1987; Dinis 1992; Jones, Kamarudin & Le
Vay 1993; Cahu, Zambonino-Infante, Quazuguel &
Le Gall 1999). Nevertheless, the potential of micro
diets is huge if properly developed, as its' com-
position can be controlled to allow for reliable
production of high-quality larvae (Buchet,
Zambonino-Infante & Cahu 2000).
Culture of tropical fishes to supply the global
aquarium trade is an important source of income
for several Asian countries (Ng & Tan 1997). The
discus (Symphysodon spp., Cichlidae) is one of the
most popular and valuable freshwater species being
intensively cultured in Malaysia, Thailand, Singa-
pore and Indonesia. Trading of this fish is character-
ized by high price and strong demand for export of
newly developed strains. Despite its commercial
value, there is a lack of research on nutritional
aspects of discus needed to develop a feeding strat-
egy for intensive culture (Chong, Hashim & Ali
2000). Discus larvae can rely solely on the bodily
mucus secretion produced by both parent fish as first
exogenous feed for around 30 days after hatch
663
Development of protease activity in discus A Chong et al.
before being separated from their parents and fed
with blood worm, Tubifex and freshly prepared wet
feed. Broodstock subjected to such intensive and
stressful larval care will, however, have shorter re-
production life spans and decreased fecundity
during the next spawning cycle. In addition, parent
fishes do not spawn until larvae are separated from
them. Because preservation of strains is a major
factor in ensuring high prices of discus, farmers
mainly feed Artemia to larvae to enable earlier sep-
aration from parent fish and to decrease the stress of
handling fry. The current high Artemia price and
low supply, competition from prawn hatcheries
and unfavourable currency exchange rate motivates
the development of dry feed to either partially or
completely replace Artemia.
The functional development of the digestive
system in growing fish larva is known to correlate
with the change from endogenous to exogenous
feeding (Ferraris, Tan & DeLa Cruz 1987; Beccaria,
Diaz, Connes & Chatain 1991). Cahu & Zambonino-
Infante (1994) also reported that enhanced digest-
ive capacity in fish larvae will improve growth and
survival. Numerous researchers have pointed to the
incompetence of digestive mechanisms in early
larvae to breakdown and assimilate formulated
diets as the main reason for poor survival and
growth (Kawai & Ikeda 1973; Kolkovski, Tandler,
Kissil & Gertler 1993; Walford & Lam 1993). Studies
on various aspects of feeding and digestion in larvae
of various species have been conducted to enable
better assessment of mircrodiets in terms of size, nutri-
ent content and stages of larvae to feed (Segner,
Rosch, Verreth & Witt 1993; Moyano, Diaz, Alarcon
& Sarasquete 1996; Cahu & Infante 1997; Ribeiro,
Sarasquete & Dinis 1999a; Ribeiro, Zambonino-
infante, Cahu & Dinis 1999). This present study
was designed to investigate changes in digestive
protease activities in developing discus larvae.
Materials and methods
Fish and sampling
Larvae were obtained from a breeding programme of
broodstock maintained at the Aquaculture Research
Complex, Universiti Sains Malaysia, Penang. After
natural pairing in a community tank, breeding was
done by placing both parents in a breeding aquar-
ium (45  45  30 cm). Commercial feed (Tetrabitt,
Tetra, Pfizer) and frozen bloodworms were fed twice
daily. A total of 15 pairs of breeding parents with
664
Aquaculture Research, 2002, 33, 663±672
histories of high fecundity and egg hatchability
were used for fry production. Newly hatched larvae
were allowed to feed on mucous substance secreted
by parent fish before Artemia was supplied 10 days
after hatching (DAH) at ad libitum thrice a day
at an estimated rate of 350±2500 naupli individ-
ual 1 day 1. The temperature of culture water
ranged from 25 to 28  C.
Larvae were sampled at 1, 3, 5, 10, 15, 20, 25,
30, 35 and 40 DAH (Fig. 1). Larvae fed with Artemia
were always sampled 2 h after feeding because pro-
tease levels have been reported to be influenced by
post-feeding duration in catfish [Clarias gariepinus
(Burchell)] larvae (Garcia-Ortega, Verreth & Segner
2000). Larvae of similar ages (0±20 DAH) were
pooled together in cold Tris-HCl (50 mm) at pH 7.5
buffer at 1 g mL 1 buffer before being hand hom-
ogenized while for late stages of larvae, dissection
was carried out and whole digestive tract sampled
and homogenized for analysis. Centrifugation at
40  C was then carried out at 10 000 g for 20 min.
Supernatant was then collected in Eppendorf tubes
and stored at 70  C prior to analysis.
Biochemical assays
The total proteolytic activity of crude enzyme ex-
tracts of each larval stage was determined using
the casein hydrolysis assay of Kunitz (1947) as
modified by Walter (1984). Assays were conducted
at pH 2.0, 7.0 and 9.0 with the use of 0.1 M of KCl-
HCl (pH 2.0) and 0.1 M of Tris-HCl (pH 7.0±9.0) as
buffers respectively (Munilla-Moran & Rey 1996;
Hidalgo, Urea & Sanz 1999). The assay was carried
out with an enzyme-substrate mixture consisting of
0.3 mL of 1% (w/v) casein in water, 0.5 mL of buffer
and 0.3 mL of crude enzyme extracts and incubated
in a water bath for 1 h at 37  C. A total of 0.5 mL of
trichloroacetic acid (TCA, 12% w/v) was then added
to the mixture to stop the reaction. This mixture was
then allowed to stand for 1 h at 4  C before centrifu-
ging at 8000 g for 15 min. Absorbance of the super-
natant was recorded at 280 nm to measure the
amount of tyrosine produced. A blank was prepared
by pre-incubating a mixture of the crude enzyme
extract, buffer and water for 1 h at 37  C, followed
by the concurrent addition of TCA and casein to
prevent any hydrolysis activities. This allows for
subtraction of absorbance caused by reagents and
especially enzyme extract due to the possible pres-
ence of materials that absorb at a wavelength simi-
lar to the product from hydrolysis (Garcia-Carreno
ß 2002 Blackwell Science Ltd, Aquaculture Research, 33, 663±672
Aquaculture Research, 2002, 33, 663±672
Figure 1 Growth of discus larvae
(dry weight) used in the experiment
from 0 to 40-DAH period. Values are
mean (mg ‡ SE) from 30 individuals.
1992). One unit of specific discus enzyme activity
was defined as the amount of enzyme needed to
produce 1 mg tyrosine min 1 mg 1 soluble protein
of enzyme extract (U mg 1 protein).
Trypsin activity was assayed using benzoyl-DL-
arginin-p-nitroanilide (BAPNA) as substrate
(Erlanger, Kokowsky & Cohen 1961). A total of
43.5 mg of BAPNA (Fluka Chemicalst) was dis-
solved in 1 mL of dimethylsulphoxide (DMSO) and
made up to 100 mL with 0.05 M of Tris-HCl buffer
containing 0.02 M of CaCl2.2H2O, pH 7.5. For assay
purposes, 25 mL of enzyme extract was mixed with
1.25 mL of freshly prepared BAPNA substrate solu-
tion and left for 10 min at 37  C before adding 30%
acetic acid to stop the reaction. The absorbance of
the resulting mixture were then determined at
410 nm followed by the calculation of trypsin ami-
dase activity (BAPNA unit mg protein 1) using the
following formula
Absorbance value at 410 nm min 1  1000  volume of reaction mixture
8800  mg protein in the assay
where 8800 is the extinction coefficient of p-
nitroaniline.
Chymotrypsin activity was assayed using succi-
nyl-(Ala)2-Pro-phe-p-nitroanilide (SAPNA) as sub-
strate (Erlanger et al. 1961). Freshly prepared
substrate comprising of 0.1 mm SAPNA in 50 mm
ß 2002 Blackwell Science Ltd, Aquaculture Research, 33, 663±672
Development of protease activity in discus A Chong et al.
Tris-HCl and 20 mN CaCl2 at pH 8.5 was used for
the assay. Next, 0.59 mL of substrate solution was
mixed with 10 mL of enzyme extract with a reaction
temperature of 25  C. An increase of absorbance
value (410 nm) was recorded every minute for
5 min. Chymotrypsin activity was then expressed
as SAPNA units mg protein 1 as
Absorbance value at 410 nm min 1  1000  volume of reaction mixture
8800  mg protein in the assay
where 8800 is the extinction coeeficient of p-nitroa-
niline.
SDS±PAGE (Laemmli 1970; Bollag & Edelstein
1991) was applied to further observe the develop-
ment of different alkaline proteases at different larval
stages with the use of zymogens for substrate
SDS±PAGE as described by Garcia-Carreno & Haard
(1993). Crude larval enzyme extract was mixed in
sample buffer (Tris-HCl 1 M pH 6.8, glycerol, SDS,
bromophenol blue) at a ratio (v/v) of 2:1. A total of
5 mL of extract/sample buffer mixture was loaded
into SDS±PAGE gels (6.0  8.0 cm) with a thickness
of 0.5 mm. The gel consisted of 5% of stacking gel
and a 12% separating gel according to Bollag
& Edelstein (1991). Electrophoresis was conducted
at 1200 V using the Mini Protean IIIt electrophor-
esis system (Bio-Rad Laboratories, Hercules, CA,
665
Development of protease activity in discus A Chong et al. Aquaculture Research, 2002, 33, 663±672

Figure 2 Specific unit activity (U mg protein 1) of enzyme extract of different discus larval stages at three different pHs
based on casein assay. Values given are mean (+ SE) from three assays. Mean with a common letter are not significantly
different (P , 0.01, Tukey's HSD).

USA) for approximately 90 min at a temperature of Results

4±6  C with electrophoresis buffer of Tris-glycine-
Figure 2 shows that the digestion rate of casein at all
sodium dodecyl sulphate. The gel was then im-
three different pHs increased with the age of larvae.
mersed in casein solution (3% in 50 mm Tris-HCl
The acidic pH curve showed low activity during the
at pH of 7.5) at 40  C for 30 min to allow absorption
first 25 days with a significant increase detected
of casein into gel. The gel with the absorbed casein
only at 30 DAH. Assays conducted at an alkaline
was then removed and placed in a water bath at
pH of 9.0 showed that alkaline type of proteases is
27  C for an additional 90 min to allow the proteases
the first type of enzymes functional in discus larvae
in the gel to digest the casein. This was followed by
with a significant increase in activity shown from 3
staining using Coomassie brillant blue (CBB) R-250
DAH. This increase continued until 10 DAH before
(Bio-Rad Laboratories, CA) dissolved in a solution
displaying a significant drop at 15±25 DAH,
containing acetic acid, methanol and distilled water
followed by significant increase again until the 40
(1 g CBB in 450 mL of methanol, 450 mL of distilled
DAH stage. As for pH 7.0, result shows that activity
water and 100 mL of acetic acid) for 60 min. The
was generally low at neutral pH for 0±40 DAH
whole gel was stained blue due to the presence of
discus larva.
casein except in areas containing protease activity,
Further assays for trypsin and chymotrypsin
which had digested the substrate. The clear bands,
showed that trypsin was detected as early as 1 DAH
indicating the presence of proteases, were more ap-
with an increase in activity until 10 DAH (Fig. 3).
parent upon destaining in methanol-acetic-acid-
There was a significant increase in activity for about
distilled water solution for an additional 90 min.
10 days before a significant rise in trypsin activity
Identification of type of proteases and its correspond-
was observed beginning from 20 DAH onward. As
ing molecular weight were based on an earlier
for chymotrypsin, very low activity was detected
discus protease characterization study (Chong,
until about 15 DAH old larva when a significant
Hashim, Lee & Ali 2002).
increase began to occur until 40 DAH, showing the
importance of this protease for latter stage larvae.
The use of the substrate SDS±PAGE method
Analysis
resulted in further findings from biochemical assays
Data were analysed statistically using analysis of (Fig. 4). Based on the presence and intensity of
variance (anova) and where significant differences various bands and earlier work on characterization
(P , 0.01) were detected, Tukey's HSD test was of each of these eight bands according to their re-
carried out to separate observed means. spective protease groups and molecular weights, we

666 ß 2002 Blackwell Science Ltd, Aquaculture Research, 33, 663±672

Aquaculture Research, 2002, 33, 663±672
Figure 3 Trypsin and chymo-
trypsin activities of different discus
larva stages according to BAPNA
and SAPNA assays (Erlanger et al.
1961). Values given are mean
from three assays with a common
letter are not significantly differ-
ent (P , 0.01, Tukey's HSD).
1 5 10 15 20 30 40 (DAH)
1,2
3,4
5,6
7,8
Figure 4 Substrate SDS±PAGE gel showing development
of eight different types of alkaline proteases at different
discus larval stages.
were able to provide estimation on the development
of each of these proteases in different larval stages
(Table 1). Trypsin and serine protease bands were
detected as early as 5 DAH extract while metallo-
protease bands occurred at 15 DAH and chymotryp-
sin at around 20±30-DAH periods.
Discussion
This study showed the late development of acidic
protease in discus larvae with significant activities
not detected until 20±25 DAH. Several studies have
reported that pepsin, the major acidic protease in
fish, has low activity levels in the early stages of
larvae (Clark, Murray & Stark 1986; Walford &
Lam 1993; Moyano et al. 1996). Kawai & Ikeda
(1973), however, reported significant pepsin
ß 2002 Blackwell Science Ltd, Aquaculture Research, 33, 663±672
Development of protease activity in discus A Chong et al.
activity in rainbow trout Oncorynchus mykiss
(Walbaum) alevins aged 2±3 days. Our observations
demonstrated that although a stomach was detected
as early as 10 DAH in discus, pepsin activity was
only significant several days later. Similar findings
were also reported in African catfish where pepsin
activity was found to increase to a functional level at
around 5±6 days after formation of the stomach
(Verreth, Torreele, Spazier, Sluiszen, Rombout,
Booms & Segner 1992). Ribeiro et al. (1999a,b)
showed that gastric glands were not formed until
27 DAH and pepsin activity was detected subse-
quently, although a stomach-like structure was pre-
sent as early as 2 days old in Senegal sole (Solea
senegalensis Kaup 1858) larvae. There was no cor-
relation between gastric gland formation and pepsin
activities in Coregonus lavaretus (Linnaeus, 1758)
larva (Segner & Rosch 1998). Segner et al. (1993)
proposed the definition of a larval `stomach' as a
structure possessing digestive glands, differentiation
of a pylorus wall, an acidic environment in the stom-
ach lumen and the occurrence of significant pepsin
activity. The low activity of pepsin in first larval stages
has also been suggested as one of the main reasons for
the failure of microformulated diets (Dabrowski
1984; Govoni, Boehlert & Watanabe 1986; Segner,
Rosch, Schmidt & von Poeppinghausen 1989; Segner
et al. 1993). Studies with African catfish revealed
that the use of exogenous feed resulted in optimum
growth only when fed to a larval stage with a func-
tional stomach despite the ability of earlier larval
stages to consume such feeds (Verreth & van
Tongeren 1989; Verreth et al. 1992). Future study
on the actual role of pepsin in protein digestion of
discus first larval stages is needed.
667
Development of protease activity in discus A Chong et al. Aquaculture Research, 2002, 33, 663±672

Table 1 Development of different classes of proteases in different stages of discus larval based on electrophoretical analysis

Days after hatching

Band* Class of proteases² Molecular weight(kDa²) 1 5 10 15 20 30 40

1 trypsin 76.5 ± ‡ ‡ ‡ ‡ ‡ ‡
2 trypsin 73.3 ± ‡ ‡ ‡ ‡ ‡ ‡
3 serine (non-trypsin/chymotrypsin) 61.4 ± ‡ ‡ ‡ ‡ ‡ ‡
4 serine (non-trypsin/chymotrypsin) 58.7 ± ‡ ‡ ‡ ‡ ‡ ‡
5 metallo 39.8 ± ± ‡ ‡ ‡ ‡ ‡
6 metallo 36.1 ± ± ‡ ‡ ‡ ‡ ‡
7 chymotrypsin 21.8 ± ± ± ± ‡ ‡ ‡
8 chymotrypsin 19.2 ± ± ± ± ‡ ‡ ‡

*Numbering of bands are based on Fig. 4.

²From Chong et al. (2002).

We observed considerable alkaline proteases ac-
tivity during the first few days of the discus larval
stage. Higher alkaline proteases activity relative to
acidic proteases in early larval stages has also been
reported for turbot, Scophtalmus maximus (Munilla-
Moran & Stark 1989), Dover sole, Solea solea (Clark
et al. 1986; Boulhic & Gabaudan 1992), Asian sea-
bass, Lates calcarifer (Walford & Lam 1993) seab-
ream, Sparus aurata (Moyano et al. 1996) Senegal
sole (Martinez, Moyano, Diaz, Alarcon & Sarasquete
1999; Ribeiro et al. 1999b) and seabass,
Dicentratchus labrax (Zambonino-Infante & Cahu
1994a). Because activity was detected before the
larvae were fed with Artemia, the contribution of
an exogenous digestive enzyme could not have
been possible. Similarly, alkaline protease was also
detected before opening of the mouth in Asian sea-
bass (Walford & Lam 1993), striped bass Morone
saxitilis (Baragi & Lovell 1986), sea bream
(Moyano et al. 1996) and sea bass (Cahu &
Zambonino-Infante 1994). Alkaline proteases have
also been detected in yolk-sac of larvae and the
glands responsible for secreting them which, prob-
ably explains their presence at first larval stages
(Kawai & Ikeda 1973; Cousin, Baudin-Laurencin &
Gabaudan 1987; Segner et al. 1989; Gawlicka,
Parent, Horn, Ross, Opstad & Torrisen 2000).
Based on BAPNA assays and electrophoresis ob-
servations, trypsin was identified as the major pro-
tease present in the first few days of developing
discus larvae. This correlates with reports from
flatfish (Ribeiro et al. 1999b), salmon, Salmo
salar L. (Dabrowski 1982), sea bream (Moyano
et al. 1996; Diaz, Moyano, Garcia-Carreno,
Alarcon & Sarasquete 1997), African catfish
(Verreth et al. 1992) and sturgeon, Acipenser fulves-
cens, Rafinesque, 1817 (Buddington 1985). Diaz
et al. (1997) also postulated that high levels of tryp-
sin in early stages of sea bream were meant to
balance the non-existence or very low activities of
pepsin. Our present study showed a significant in-
crease in trypsin in 1±10 DAH stage larvae followed
by a decrease for several days. This observation has
also been reported by numerous researchers (Kawai
& Ikeda 1973; Pedersen 1993; Walford & Lam
1993; Cahu & Zambonino-Infante 1994;
Zambonino-Infante & Cahu 1994b; Diaz et al.
1997; Ribeiro et al. 1999b). Govoni et al. (1986)
suggested that the early rise in trypsin activity
could be due to an exogenous contribution during
feeding of live prey while the latter increase could be
due to actual secretion from a newly formed pan-
creas structure. Factors such as decrease in synthe-
sis rate coupled with increase in denaturation of
trypsin due to formation of the stomach and the
need for an acidic environment for pepsin activities
which may result in denaturization of trypsin activ-
ity have been suggested as possible reasons for the
drop in trypsin levels observed in intermediate
stages larvae (Lauff & Hofer 1984; Pedersen,
Nilssen & Hjelmand 1987; Zambonino & Cahu
1994b). Walford & Lam (1993), for instance,
reported a correlation between the decrease of tryp-
sin activity with the increase in pepsin activity in
Asian sea bass larvae. In addition, they also sug-
gested that homogenization of whole larval body
during preparation of crude larval enzyme extract
could have released natural occurring protease in-
hibitors present in muscles and serum. Moyano et al.
(1996) concluded that the use of enzyme activity

668 ß 2002 Blackwell Science Ltd, Aquaculture Research, 33, 663±672

Aquaculture Research, 2002, 33, 663±672
per unit protein as a measurement of enzyme activ-
ity could cause irregularities in measurements as the
whole-body protein content on larvae changes dras-
tically during development. These suggestions prob-
ably explain the increase of enzyme activity values
at 20 DAH onwards as crude enzyme extract was
obtained directly from the digestive tract of larvae
instead of whole body homogenate from this stage
onwards.
Our study also the showed a late development of
chymotrypsin as compared with trypsin. This has
also been reported in several other freshwater and
marine species (Lauff & Hofer 1984; Moyano et al.
1996; Diaz et al. 1997). An electrophoresis gel
image showed a significant increase in metallo-pro-
teases activity from 25 DAH onward. In comparison,
this group of proteases also showed lower activity
than the serine proteases group as also reported
elsewhere (Lauff & Hofer 1984; Clark et al. 1986).
The development of various functional digestive
phases in discus larval in comparison with several
other fish species is shown in Fig. 5. Mouth opening
in discus larval occurs later as compared with sev-
eral marine species such as sole and Asian sea bass.
Freshwater larvae usually have a larger supply of
yolk-sacs in comparison with marine species, there-
fore enabling them to delay on exogenous feeding
until much later stages (Sorgeloos & Leger 1992).
Gut differentiation from a straight tube to a folded
alimentary tract also happened at this phase. The
presence of alkaline proteases was found as early as
has been found in several other species. Based on
this observation together with other important
events such as a high activity of pepsin and a func-
Discus
Sea bream
Sole
Striped bass
Seabass
Asian sea AKP, MO Y,D
bass
Days after
hatching 1-3
AKP:
ACP:
DR:
MO:
Figure 5 Comparison of digestive S:
system and protease development of C:
Y:
discus larvae with other studies.
ß 2002 Blackwell Science Ltd, Aquaculture Research, 33, 663±672
Development of protease activity in discus A Chong et al.
tional stomach, we estimate that development of a
full functioning digestive system in discus larva
occurs at about 40 DAH period.
The feeding strategy of fish larvae during the
nursery phase will depend on information about
digestive system development. Morphological indi-
cators, such as the beginning of mouth and anal
opening, formation of a stomach, intestine and
other organs with digestive functions such a pan-
creas and liver have been suggested (Dabrowski &
Culver 1991; Segner et al. 1993; Ribeiro et al.
1999a). Biochemical cues in terms of presence and
level of certain proteases are also regarded as vital as
the presence of certain digestive organs might not
correlate with ability of larva to receive microdiets
due to limited capacity of digestive enzymes to break
down nutrients (Lauff & Hofer 1984; Walford & Lam
1993; Ribeiro et al. 1999b). However, some re-
searchers concluded that despite the availability of
these cues, the main objective of larval feeding
should be provision of easily digested and assimi-
lated nutrients whether through use of live prey,
micro diets or a combination of both (Mitchell,
Nickum & Long 1986; Holt 1992; Verreth et al.
1992; Person-Le Ruyet et al. 1993; Kolkovski,
Tandler & Izquierdo 1997).
The current practice of relying on Artemia alone
as feed of discus larval from 7 DAH until 30±40
DAH would be uneconomical as the price of
Artemia continue to increase. Various factors will
have to be considered to ensure similar survival
and growth performances of larvae if microdiets
were to replace Artemia. The role of Artemia in con-
tributing exogenous digestive enzymes has been
AKP Y, D, MO ACP, S C Present study;
Chong (2000)
AKP Y, MO C Moyano et al.
(1996)
MO, D, AKP S C Ribeiro et al.
(1999a,b)
AKP MO S Baragi & Lovell
(1986)
AKP MO S Cahu &
Zambonino (1994)
S C Walford & Lam
(1993)
3-5 5-10 10-20 20-30 30-40 > 40
alkaline protease activity (serine protease, trypsin, chymotrypsin)
acidic protease activity (pepsin)
Gut differentiation
Mouth open
Formation of functional stomach
Completion of digestive system morphologically with digestive enzymes
yolk adsorption complete
669
Development of protease activity in discus A Chong et al.
reported in larvae of turbot (Munilla-Moran, Stark &
Barbour 1990) and whitefish, Coregonus sp. (Lauff &
Hofer 1984). Serine proteases, for example, have
been identified in Artemia (Diaz et al. 1997; Warner
& Matheson 1998). In contrast, several later reports
have quantified a very low direct contribution of
dietary enzyme by ingested Artemia and suggested
that increased digestive capacities in larvae fed live
feeds are the result of pancreatic and intestinal de-
velopment instead (Cahu & Zambonino-Infante
1994; Moyano et al. 1996; Zambonino Infante,
Cahu, Peres, Quazuguel & Le Gall 1996;
Kurokawa, Shiraishi & Suzuki 1998). Based on the
present study, complete formation of a stomach and
intestine occurred together with high activities of
several proteases beginning at 25 DAH. The use of
a dry diet could therefore start from this larval phase
to ensure full capacity of digestion and adsorption.
Although the high levels of alkaline proteases
detected in the first 20 days after hatching may
suggest the possibility of microdiet usage at earlier
stages, other factors such as composition of diet and
weaning period should be taken into consideration.
Poor growth and survival, for instance, were
reported in sea bass larvae fed a microdiet as com-
pared with Artemia despite showing high levels of
protease secretion (Cahu & Zambonino-Infante
1997). Several strategies such as co-feeding of a
dry diet with live food (Canavate & Fernandez-Diaz
1999), use of nutritional factors such as lipids and
free amino acids isolated from live food (Kolkovski,
Koven & Tandler 1997; Koven, Kolkovski, Hadas,
Gamsiz & Tandler 2001), supplementation of dietary
enzymes (Koven, Kolkovski, Tandler, Kissil & Sklan
1993) and use of various protein hydrolysate (Cahu
et al. 1999; Kolkovski & Tandler 2000), however,
could possibly enable replacement of Artemia in
discus larval nutrition at even earlier stage.
Acknowledgments
We appreciate Universiti Sains Malaysia for funding
of this study through a short-term research fund
(PBIOLOGI 622093).
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