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