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

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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 Our unpublished results shows that early stages discus larva requires parental mucus to undergo normal growth as compared to latter stages where exogenous live feed such as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. 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