Characterisation of proteins in epidermal mucus of discus fish ...

2 K. Chong et al. / Aquaculture xx (2005) xxx-xxx
problems of inadequate growth and large scaled
mortality occur. Both discus parents display
advanced behavior of parental care where freeswimming
larvae feed on epidermal mucus secretion
produced by both male and female brood. This
natural method is widely utilized in discus hatcheries
to obtain healthier and higher quality fry. However,
extended period of larval care by discus parents often
negatively affect their subsequent reproductive performances.
This is due to the stressful nature of
having to constantly replace new mucus layer for
protection and osmoregulatory functions. An important
goal in discus culture is to develop reliable larva
feed that can either completely or partially replace
parental mucus. An understanding on the biochemical
composition of parental mucus will provide
useful insights to aid the development of such feed.
In this present paper, we reported a study conducted
to investigate the protein properties of discus fish
mucus.
2. Material and methods
2.1. Discus breeding
Broodstock used for experiments were selected
from stock population maintained at Laboratory of
Fish Biology, Universiti Sains Malaysia. Parental
fishes ready for breeding are easily recognized from
their aggressive territorial behavior. Individual pairs
are separated into breeding tanks (2' x2' x 1.5')
respectively. Successful spawning will be followed
by a 3-4 days period of egg incubation prior to
hatching. These batches of parent-fry were then used
for different types of experiments outlined below. All
parents were fed with frozen bloodworm and wet
paste consisting of minced beef-heart and shrimp
throughout the experimental period.
2.2. Larval biting rate
Ontogenic feeding behavior of discus larvae on
parental mucus secretion was observed through
determination of its biting rate. The numbers of bites
per 30 s by larvae on parental mucus was recorded
from six randomly selected larvae at selected freeswimming
days at 1200 hours. For each individual
larva, a total of three counts were carried out.
Comparisons were made between
i. larval feeding solely on mucus.
ii. larval feeding on mucus with supplementation of
freshly hatched Artemia nauplii. Counts were carried
out at 1 and 3 h after Artemia supplementation.
2.3. Mucus sampling
Fish mucus was sampled from female parental
discus (600-700 g) performing parental care on day
10-15 of free-swimming larvae. Mucus was also
sampled from juvenile discus aged 5-6 months (350­
400 g). Briefly, collection was done through very
gentle scrapping of the dorsal-lateral part of body
with clean spatula to stimulate production of a fresh
mucus layer. A clean glass pipette was used to collect
the mucus followed by immediate transfer to clean
glass vials on ice. Sampling was not done at the
ventral area to avoid possible urinal contamination.
Collected mucus was then centrifuged at 13,200 xg
for 20 min at 4 °C followed by storage of supernatant
in -70°C prior to analysis.
2.4. Biochemical analysis
2.4.1. Protein content
Mucus protein content was analyzed using the
Bradford Assay (Bradford, 1976). Briefly, 10 III of
supernatant for each mucus sample was mixed with
200 III of the BIORAD® assay kit reagent and
allowed to stand for 15 min. Absorbance value was
recorded at 595 nm followed by determination of
protein concentration from a standard protein concefltration-absorbance
curve.
2.4.2. SDS-PAGE
SDS-PAGE (Laemmli, 1970) was also used to
separate mucus protein from both parental and
juvenile stages. Mucus supernatant was mixed
with sample buffer (Tris-HCI 1 M pH 6.8,
glycerol, SDS, bromophenol blue, 2 mercapthoethanol)
at a ratio of 4: 1 (v Iv). A total of 24 III of
this mixture corresponding to a protein loading of
13-18 Ilg was loaded into gels (6.0 x 8.0 em,
thickness 0.75 mm). Electrophoresis was conducted
at 120 V using the Mini Protean IIJ® electrophoresis

system (BIORAD® Laboratories, California) for
approximately 120 min. This was followed by
staining using Coomassie Brillant Blue R-250 (BIO­
RAD®) dissolved in acetic acid, methanol and
distilled water for 120 min followed by overnight
destaining in a solution of methanol, acetic acid and
distilled water. Broad Range (BIORAD®) molecular
weight marker ranging from 14.4 kDa to 200 kDa
was also used for molecular weight estimation. Gels
were analyzed using the QUANTITY ONE® gel
analysis software (BIORAD®). Wet blotting process
was also carried out on SDS gel after completion of
electrophoresis using a PVDF membrane on a Mini
Trans Blotter (BIORAD®). The band of interest was
then excised from the membrane, subjected to
trypsin digestion, followed by mass spectrometry
analysis using the Quadrupole-Time of Flight
(MICROMASS Q-TOF®) analysis. Obtained peptide
mass fingerprints were then subjected to database
search using the MASCOT protein database (Perkins
et aI., 1999).
2.4.3. Free amino acids
Free amino acid content of discus mucus was
analyzed using the preparation of PTC derivatives of
amino acids using the procedure described by
Bidlingmeyer et al. (1984). Briefly, stock solutions
of each amino acid standard at concentrations of 250
Ilmol/ml were prepared in distilled water. A total of 10
III aliquots of the standard amino acid stock solution
was then added to 20 III of ethanol-water-TEA
(2: 2 : 1) mixture, prepared fresh daily in a small glass
vial and then shaken thoroughly. The mixture was
then dried under vacuum by using an Edward Model
E2M8 Vacuum pump (Sussex, England).
The derivatization mixture consisting of ethanol­
TEA-water-PITC (7: 1: 1: 1) was also prepared fresh
daily. An aliquot of 20 III of the freshly prepared
derivatization mixture stated above was added to each
dried sample, shaken thoroughly and allowed to stand
at room temperature for 20 min. Excess reagent and
by-products were removed under vacuum. The PTCamino
acids were then stored at - 20°C until use. An
aliquot of 200 III of trichloroacetic acid, TCA (40%)
was added to an equal volume of mucus supernatant.
The mixture was shaken thoroughly and then allowed
to incubate at 4 °c for 1 h followed by centrifugation
for 30 min to sediment down the protein precipitate.
K. Chong el al. I Aquaculture xx (2005) xxx-xxx 3
The supernatant, around pH 1, was carefully decanted
into a glass vial and the contents freeze-dtied. This
freeze-dried sample was then reconstituted in 20 III of
distilled water followed by derivatization as described
above. The derivatized samples were then reconstituted
in 200 III of mobile phase B. As for the blank,
sample was just dissolved in 200 III ofmobile phase B
without derivatization. This is to ensure there are no
ultraviolet absorbing compounds present in the
sample, which might interfere in the detection of free
amino acids in the slime.
The HPLC equipment consisted of a solvent
delivery system comprising a Gilson Model 305 main
pump (Villiers Ie Bel, France) and a Gilson Model 302
secondary pump, a Gilson Model 802 C manometric
module, a Gilson Model 811B dynamic mixer, Gilson
Model 302 and Model 305 pumps, Rheodyne 7725
injector (Cotati, CA, USA), a 20 f.ll sample loop, a 15
cm x 4.6 mm ID (5 f.lm) Supelcosil LC18 column
(Bellefonte, PA, USA) and guard column thermostated
at 30°C, a Spectra-physics UV2000 ultraviolet
detector (San Jose, CA, USA) and a Shimadzu
integrator-plotter (Tokyo, Japan). For the elution of
the PTC amino acids, a gradient profile was developed
to optimize a complete separation of the PTC
amino acid derivatives. The solvent system consisted
of two eluents namely solvent A (acetate buffer, pH
6.35,0.14 M containing 0.5 mUI TEA) and solvent B
(60% acetonitrile in water). The total flow rate used
was at 1 mUmin. The washing step at 100% B was
programmed to run for 30 min to wash away any
residual contaminants that could be present in the
sample. The system was returned to 100% A for the
next injection.
3. Results
Throughout experimental period, discus larvae
showed close contact with parents, feeding on their
epidermal mucus. Fig. I shows a gradual increase in
this biting rate from first free-swimming day till day
12-15. Biting rate then decreased gradually as larvae
developed. In the presence of Artemia however,
mucus biting rate also dropped at 1 h after supple-'
mentation of Artemia as larvae were either still
actively preying on nauplii or showed decrease in
feeding due to filled gut. Biting rate increased

K. Chong et al. / Aquacullllre xx (2005) xu-xxx
Table 1
Peptide fragments positively identified utilizing the MASCOT search on band Z
Observed Mr (expt) Mr (calcu)
827.41
906.45
973.51
1153.57
1180.54
1200.63
1348.67
1432.74
1443.70
826.40
905.46
972.52
1152.60
1179.58
1199.66
1347.62
1431.77
1442.74
826.42
905.46
972.39
t 152.55
1179.56
1199.65
1347.68
1431.74
1442.70
Peptide
5
FASFIDK
FLEQQNK
QLDGLGNEK
EYQELMNVK
NMQGLVEDFK
IKDLEDALQR
WSLLQDQTITR
VDALQDEINFLR
ANLEAQIAEAEER
I MSFSRTSYTSGGGGGMGGGTISIRRSFTSQSMIAPGSTRMSSGSVRRSGA
51 GFGGGMGMGGGSGGGFSYISSSSAGGYGGGLGAGFGGGMGGGYPITAVTI
101 NQSLLAPLNLEIDPNIQVVRTHEKEQIKTLNNRlMUIJjHvRIII"llIij9M
151 LETKW"'IIUIUiiiij

6 K. Chong et al. / Aquaculture xx (2005) xxx-xxx
the proteins showing higher expression in parental
mucus as a type II epidermal keratin.
An increase in contact behavior between discus fry
and parental mucus was observed until the first 12
free-swimming days before showing a decreasing
trend. This behavior correlates observations recorded
in C. citrinellum, another cichlid species utilizing
mucus secretion as larval feed. (Noakes and Barlow,
1973; Schutz and Barlow, 1997). The lower biting rate
after feeding on Artemia indicates that intake of
parental mucus is influenced by hunger, as reported in
C. citronellum fries (Schutz and Barlow, 1997). Our
unpublished results shows that early stages discus
larva requires parental mucus to undergo normal
growth as compared to latter stages where exogenous
live feed such as Artemia can be utilized to replace
parental mucus. It is also worth noting that unlike C.
citronellum, feeding of parental secretion is nonobligatory
in discus fry where mass mortality have
been reported in fry isolated from parents (Hildemann,
1959). Our previous study revealed that a significant
increase in the activities ofall vital digestive enzymes
occurred during the 15-20 free-swimming days in
discus larvae (Chong et aI., 2002). The lack of a fully
functional digestion system in earlier larval stages
probably explains the need to rely on parental
mucosal secretion. Furthermore, mucosal secretion is
an effective method to transfer important substances
such as hormones and antibodies to enhance survival
and growth performances in developing larvae (Kishida
and Specker, 2000).
Our study showed higher total protein content in
parental mucus as compared to juveniles. There have
been several reports on changes in fish mucus protein
content and composition as results of several physiological
related conditions such as maturation, smoltification
and starvation (Saglio and Fauconneau,
1985a; Heming and Paleczny, 1987; Ottesen and
Olafsen, 1997; Fagan et aI., 2003). Schutz and Barlow
(1997) also reported a fluctuating trend in parental C.
citronellum mucus protein content throughout the
larval raising period. The higher protein content in
discus parental mucus obtained here could directly or
indirectly relate to larval feeding activities. We do not
rule out the possibility that the increased protein
concentration could be due to disruption of epidermal
cells and hence, leaking of cellular and plasma
proteins into the mucus. Consequently, it is also
possible that the stressful condition of losing epidermal
layers of mucus and cells initiated physiological
stress-related responses that resulted in elevated
protein contents. Elsewhere, various stress-related
factors such as handling, presence of abiotic contaminants
and pathogenic infections have reportedly
caused elevation of mucus protein content (Pickering
and Macey, 1977; Ross et aI., 2000; Lebedva et aI.,
2002). There is still a lack of understanding on the
actual regulatory mechanisms of mucus protein
expression under the above-described conditions.
Several studies have also pointed out that such
regulation exists at the transcription level involving
a widespread family of genes (Sakamoto et aI., 2002;
Lindenstrom et aI., 2003). Prolactin, a hormone
known to mediate parental behavior in higher
vertebrates have also been reported to induce proliferation
of mucus-producing goblet cells for osmoregulatory
control in several fish species, including
discus (Blum and Fiedler, 1965; Ogawa, 1970;
Manzon, 2002). Hildemann (1959) reported presence
of larger-sized and hypertrophied goblet cells in
discus parent epidermal skin as compared to nonparental
fish to accommodate increased production of
mucus for larval rearing.
The existence of a large number of proteins
separated by molecular weight in discus mucus shown
in this study points to the importance of discus
epidermal mucus for a multi-array of functions.
Depending on the origin and function, mucus in
general contains water, glycoproteins, secretory proteins,
amino acids, lipids, sloughed epithelial cells and
bacterias (Shephard, 1994). Mass spectrometry analysis
on one of the band showing higher expression in
discus parental mucus revealed an identity of a
epidermal type II keratin. Among the major function
for this subfamily of keratin is epidermis architecture
and regulation ofepidermal cell development (Conrad
et aI., 1998). The detection of this structural protein in
discus mucus however, remains unclear. One possible
explanation could be due to leaking of cellular and
plasma proteins in the mucus itself due to constant
nipping by fry. Although not detected here, other
studies have pointed to the existence of important
larval growth-related substance in parental mucus.
Several forms ofvitellogenin were identified in mucus
of female tilapia, although their role as source of
nutrient to developing fry remains unconfirmed

(Kishida and Specker, 1994, 2000; Takemura, 1994).
Schutz and Barlow (1997) isolated growth-promoting
prolactin and thyroid hormones from mucus ofMidas
cichlid. In tilapia, it has been suggested that transfer of
antibodies occurs during parental feeding oflarvae via
mucus secretion (Mol' and Avtalion, 1990; Takemura
and Takano, 1997).
This present study also demonstrated the presence
of various free amino acids in mucus. In addition to
their role as substrates for protein synthesis, amino
acids are also vital as neurotransmitters and osmoregulators.
All ten indispensable amino acids were
found present in both parental and juvenile mucus of
this species. Several indispensable amino acids such
as lysine, isoleucine and phenylalanine were present at
high levels in discus mucus. Saglio and Fauconneau
(1985b) also reported relatively higher content of
lysine as compared to other amino acids in gold fish
(Carrasius auratus) mucus. In developing marine
larvae with immature digestive systems, adsorption of
free amino acids provides a critical source of energy
and nutrient as compared to peptides and proteins
(Ronnestad et al., 2003). The importance of mucosal
free amino acids as source of nutrient for discus fry
remains to be studied.
This present study proposed the possible existence
and ability to upregulate vital substances needed by
developing larvae in discus parental mucus. Efforts to
further isolate and characterize parental mucus using
higher end tools such as 2-D electrophoresis and mass
spectrometry are currently being undertaken. These
tools will enable better separation of proteins, to
increase the possibility of identifying proteins that
may have an important role towards survival and
growth of discus larvae.
Acknowledgements
We wish to thank the Ministry of Science,
Technology and Environment ofMalaysia for funding
of this project under Project No. 09-02-05-1069 EA
001. Appreciation also goes to Malaysian Toray
Scientific Foundation for award of a research grant
(2001-2002) to partially fund this work. The assistance
of Dr. Shashikant 1. and Ms. Wang X.H. at the
Department of Biological Sciences, National University
of Singapore are also acknowledged.
K. Chong et al. / AquaculllIre xx (2005) xxx-xxx 7
References
Aranishi, F., Nakane, M., 1997. Epidennal proteinases in the
European eel. Physiol. Zool. 70 (5), 563-570.
Bidlingmeyer, BA, Cohen, SA, Tarvin, T.L., 1984. Rapid analysis
of amino acids using pre-column derivatisation. J. Chromatogr.
336, 93-104.
Blum, v., Fiedler, K., 1965. Honnonal control ofsome reproductive
behaviour in some cichlid fish. Gen. Compo Endocrinol. 5,
186-196.
Bradford, M., 1976. A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing the
principle of protein dye-binding. Anal. Biochem. 72, 248.
Chong, A., Hashim, R., Lee, L.C., Ali, A., 2002. Characterisation of
protease activity in developing discus Symphysodon aeqllifasciata
larva. Aquac. Res. 33, 663-672.
Conrad, M., Lemb, K., Schubert, T., Mnrkl, J., 1998. Biochemical
identification and tissue-specific expression patterns of
keratins in the zebrafish Danio rerio. Cell Tissue Res. 293,
195-205.
Fagan, M.S., O'Byrne-Ring, N., Ryan, R., Cotter, D., Whelan, K.,
Mac Evilly, U., 2003. A biochemical study ofmucus lysozyme,
proteins and plasma thyroxine ofAtlantic salmon (Salrno salar)
during smoltification. Aquaculture 222, 287 - 300.
Handy, R.D., Eddy, F.B., Romain, G., 1989. In vitro evidence for
the ionoregulatory role of rainbow trout mucus in acid, acidJ
aluminium and zinc toxicity. 1. Fish BioI. 35,737-747.
Heming, T.A., Paleczny, E.J., 1987. Compositional changes in skin
mucus and blood serum during starvation of trout. Aquaculture
66,265-273.
Hildemann, W.H., 1959. A cichlid fish, Symphysodon discus with
unique nurture habits. Am. Nat. 93, 27-34.
Kishida, M., Specker, J.L., 1994. Vitellogenin in the surface mucus
oftilapia (Oreochrornis mossarnbiclls): possibility for uptake by
the free-swimming embryos. J. Exp. ZooI. 268, 258-268.
Kishida, M., Specker, J.L., 2000. Paternal mouthbrooding in the
black chinned tilapia, Sarotherodon melanotheron (Pisces:
cichlidae): changes in gonadal steroids and potential for
vitellogenin transfer to larvae. Honn. Bo::hav. 37,40-48.
Laemmli, U.K., 1970. Cleavage of structural proteins during
assembly of the head of bacteriophage T 4 . Nature 227,
680-685.
Lebedva, N., Vosyliene, M.Z., Golovkina, T., 2002. The effect of
toxic and heliophysical factors on the biochemical parameters of
the external mucus of carp (Cyprinus carpio L.). Arch. Pol.
Fish. 10,5-14.
Lindenstrom, T., Buchman, K., Secombes, C.J., 2003. Gyrodactyills
deljavini infection elicits IL-I beta expression in rainbow trout
skin. Fish Shellfish Immunol. 15, 107-115.
Manzon, L., 2002. The role of prolactin in fish osmoregulation: a
review. Gen. Compo Endocrinol. 125,291-310.
Mor, A., Avtalion, R.R., 1990. Transfer of antibody activity from
immunized mother to embryos in tilapias. J. Fish BioI. 37,
249-255.
Noakes, D.L.G., 1979. Parent-touching behaviour by young
fishes: incidence, function and causation. Environ. BioI. Fish.
4,389-400.

8 K. Chong et al. / Aquaculture xx (2005) xxx-xxx
Noakes, D.L.G., Barlow, G.w., 1973. Ontogeny of parent-contacting
behaviour in young Cichlasoma citrinellum (Pisces Cichlidae).
Behaviour 46, 221-255.
Ogawa, M., 1970. Effects of prolactin on the epidermal mucous
cells of the goldfish Carassius auralus L. Can. J. Zoo!. 48,
501-503.
Ottesen, O.H., Olafsen, l.A., 1997. Ontogenetic development and
composition of the mucuos cells and the occurrence of saccular
cells in the epidermis of Atlantic halibut. l. Fish Bio!. 50,
620-633.
Perkins, D.N., Pappin, D.J., Creasy, D.M., Cottrell, l.S., 1999.
Probability-based protein identification by searching sequence
databases using mass spectrometry data. Electrophoresis 20
(18),3551-3567.
Pickering, A.D., Macey, D.J., 1977. Structure, histochemistry and
the effect of handling on mucous cells of the epidennis of the
char SalvelillUs alpinus. l. Fish Bio!. 10,505-512.
Ronnestad, 1., Tonheim, S.K., Fyhn, H.J., Rojas-Garcia, c.R.,
Kamisaka, W., Koven, w., Finn, R.N., Terjesen, B.F., Barr, Y.,
Conceicao, L.E.C., 2003. The supply of amino acids during
early feeding stages of marine fish larvae: a review of recent
findings. Aquaculture 227, 147-164.
Ross, N.W., Firth, KJ., Wang, A.P., Burka, J.F., Johnson, S.c.,
2000. Changes in hydrolytic enzyme activities of na'ive Atlantic
salmon Sa/mo sa/ar skin mucus due to infection with the
salmon louse Lepeophtheirlls salmonis and cortisol implantation.
Dis. Aquat. Org. 41, 43-51.
Saglio, P., Fauconneau, B., 1985a. Free amino acid content in fish
skin mucus as an indicator ofstarvation: a preliminary report. 1.
Fish Bio!. 9, 537-541.
Saglio, P., Fauconneau, B., 1985b. Free amino acid content in the
skin mucus of goldfish, Carrasius aurallls L.: influence of
feeding. Compo Biochem. Physio!. 82A, 67 - 70.
Sakamoto, T., Yasunaga, H., Yokota, S., Ando, M., 2002. Differential
display of skin mRNAs regulated environmental conditions
in a mudskipper. l. Compo Physio!., B 172,447-453.
Schutz, M.S., Barlow, G.w., 1997. Young of the Midas cichlid get
biologically active nonnutrients by eating mucus fonn the
surface of their parents. Fish Physio!. Biochem. 16, 11-18.
Shephard, K.L., 1994. Functions for fish mucus. Rev. Fish Bio!.
Fish. 4, 401-429.
Sundara. R.B., 1962. The extraordinary feeding habits ofthe catfish
Mystus aor (Hamilton) and MysIlIs seengha/a (Sykes). Proc.
Nat!. Acad. Sci., India 28, 193-200.
Takemura, A., 1994. Vitellogenin-like substance in the skin mucus of
tilapia Oreochromis mossambicus. Fish. Sci. 60 (6), 789-790.
Takemura, A., Takano, K., 1997. Transfer of maternally-derived
immunoglobin (IgM) to larvae in tilapia Oreochromis 1IlossambiclIs.
Fish Shellfish Immuno!. 7, 355-363.