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Research Article

Hemolytic and Antibacterial Studies on Skin Mucus of Eel Fish, Anguilla anguilla Linnaues, 1758

S. Bragadeeswaran and S. Thangaraj
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Eel fish, Anguilla anguilla skin contains specialised glandular cells that produce mucins and substance with antimicrobial and noxious properties. In the present study, Anguilla anguilla was collected from Parangipattai coastal waters of Tamil Nadu, India. The crude and aqueous extract of mucus was studied for hemolytic assay on chicken blood and goat blood, antibacterial activity against shrimp culture pond pathogens viz., Pseudomonas aeruginosa, Vibrio parahemolyticus, Escherichia coli, Staphylococcus aerius, Salmonella typhi, Salmonella paratyphi, Klebsiella pneumonia, Klebsiella oxytoca, Proteous microbilus and Lactobacillus vulgaris. The maximum specific hemolytic unit of 16 HU was recorded in chicken blood and minimum of 2 HU against goat blood. Whereas the antibacterial activity, maximum zone of inhibition 10 mm and minimum of 1 mm were showed in Salmonella paratyphi and Staphylococcus aerius, respectively. This present study has revealed that preliminary assays indicated that eel fish mucus was an interesting source for hemolytic and antibacterial activity.

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S. Bragadeeswaran and S. Thangaraj, 2011. Hemolytic and Antibacterial Studies on Skin Mucus of Eel Fish, Anguilla anguilla Linnaues, 1758. Asian Journal of Biological Sciences, 4: 272-276.

DOI: 10.3923/ajbs.2011.272.276

Received: March 08, 2010; Accepted: June 05, 2010; Published: October 13, 2010


All living organisms including fish coexist with a wide range of pathogenic and nonpathogenic microorganisms and, therefore, possess complex defense mechanisms which contribute to their survival. The mechanism is innate immune system that combats pathogens from the moment of their first contact (Kimbrell and Beutler, 2001). The eel A. anguilla is the most abundant of the genus of Anguilla. The epidermal layer of fishes are contains specialised glandular cells that produce mucins and alarm substance (Smith, 1992). These substances having potential of antimicrobial and noxious properties (Knouft et al., 2003). Skin club cells or sacciform cells of the eel fishes have produced noxious substances that exhibit very lesser toxicity to higher venomous activity (Mittal et al., 1981). The mucus gives the physical production of the skin and also serves as an anti-predator role in fishes (Fishelson, 1996). Mucus products the skin from pathogens and suspended particles (Knouft et al., 2003). The epidermal; mucus is advantages not only to fish but also in mankind for various purposes like defense, cultural ponds. Only the very few studies are available in the crinotoxin studies in fish mucus.

Mucus is the slimy secretion consisting of mucins and combination of other substances such as inorganic salts immunoglobulin and lipids suspended in water giving it characteristic lubricating properties (Pearson and Brownlee, 2005). The functional properties of mucus depend on its capacity to form a gel on the epithelial surface. This gel-forming property is controlled by the amount, size and degree of cross-linking present between the mucus glycoprotein (mucins) (Smith, 2002). Mucins are filamentous, high molecular weight glycol-proteins (0.5-20 MD). There are two major forms of mucins known in humans-monomeric and oligomeric. The monomeric form is generally present on the cell surface. Where as the oligomeric mucin has been held responsible for the gel like properties of secreted mucin (Thornton and Sheehan, 2004). The present study was carried out the hemolytic and antimicrobial activity from skin mucus of eel fish A. anguilla.


Collection and preparation of crude extract: This study was conceded out during December to March 2008. The eel fish, A. anguilla was collected from Annankovil landing centre, Parangipattai, (lat. 11°26' N long. 79°49' E) Tamil Nadu, Southeast coast of India. Fish immediately brought to the laboratory in fresh condition for mucus collection. The mucus was centrifuged at 15,000 rpm for 15 min. The supernatant was collected and lyophilized. Lyophilised powder was stored at 4°C for further analysis.

Haemolytic study
Preparation of erythrocytes suspension: The crude extract of A. anguilla was assayed on chicken and goat blood followed by the method of Paniprasad and Venkateshvaran (1997). The chicken and goat blood were obtained from the nearby slaughter house in Parangipettai and was added with 5% of ethylenediaminetetraacetic (EDTA) solution as an anticoagulant of blood. The blood samples were centrifuged thrice at 5,000 rpm for 5 min at along with phosphate buffer saline (pH 7.4) about double the quantity of blood. The supernatant was discarded. About 1.0 mL of packed RBC thus obtained was resuspended in phosphate buffer saline to obtain a 1% RBC suspension used for hemolysis study.

Haemolytic assay-microtitre pate method: Haemolytic assay was performed in V shaped sterile Laxbro microtitre plate. Serial two fold dilutions of the crude extract (100 μL; 1 mg crude in 1 mL PBS) were made in PBS (pH 7.2) starting from 1: 2. An equal volume of 1% human RBC was added to each well. The plate was shaken for mixing the RBC and crude extract. The plates were incubated at room temperature for 2 h before reading the results. Appropriate control was included in the tests. Erythrocytes suspension to which distilled water was added (100 μL, respectively) served as blanks as negative control. Formation of the button in the wells was taken as negative. Reciprocal of the highest dilution of the venom extracted showing the haemolysis was taken as one haemolytic unit.

Antibacterial activity: Antibacterial activity was carried out by using the standard disc diffusion method (Rajaganapathi, 1996; Murugan and Santhanaramasamy, 2003) against Pseudomonas aeruginosa, Vibrio parahemolyticus, Escherichia coli, Staphylococcus aerius, Salmonella typhi, Salmonella paratyphi, Klebsiella pneumonia, Klebsiella oxytoca, Proteous microbilus and Lactobacillus vulgaris. The extracts were applied to 6 mm sterile discs in aliquots of 30 μL of crude extract, allowed to dry at room temperature and placed on agar plates seeded with microorganisms. The bacteria were maintained on nutrient agar plates and incubated at 37°C for 24 h. Zones of growth inhibitions were measured by scale.


All blood groups were showed good activity. The crude extract of A. anguilla was showed the maximum hemolytic unit recorded in chicken blood (16 HU) and minimum of 2 HU was recorded in goat blood.

Image for - Hemolytic and Antibacterial Studies on Skin Mucus of Eel Fish, Anguilla anguilla Linnaues, 1758
Fig. 1: Antibacterial activity of eel fish skin mucus against shrimp culture pond pathogens

Hemolysis of human and sheep red blood cells has been studied by Al-Hassan et al. (1982). In earlier reported by Al-Lahham et al. (1987) has studied the specific activity of the catfish epidermal factor is 20.6 units mg-1 protein, a level somewhat lower than those of most protein hemolytic factors. In the present study also had seen good activity against blood samples. Whereas the antibacterial activity the maximum inhibition zone (10 mm) was showed against salmonella paratyphi in the crude extract of A. anguilla and minimum of zone inhibition (1 mm) was recorded against Staphylococcus aerius in the aqueous extract shown in Fig. 1. Subramanian et al. (2008) reported the acidic epidermal mucus extract of hagfish exhibited antimicrobial activity against several fish and human pathogens. The aqueous extracted mucus of the examined fish species having the presence of various innate antimicrobial agents such as lysozyme, cathepsin B and trypsin-like proteases was demonstrated by Subramanian et al. (2007). Smith et al. (2000) have reported the bacteriolytic activity against Micrococcus lysodeikticus, Pasteurella piscicida, L. anguillarum and Micrococcus luteus from skin mucus of common carp C. carpio and rainbow trout O. mykiss. In this present study also exhibited good antimicrobial activity against several pathogens.

Fish have adapted to survive in pathogen-rich aquatic environment. Their primary protection against invading pathogens is the epidermal mucus which contains a variety of antimicrobial components such as AMPs, lysozyme, proteases and lectins (Ellis, 2001). The antimicrobial property of epidermal mucus against infectious pathogens has been demonstrated previously in rainbow trout O. mykiss (Austin and McIntosh, 1988), ayu (Plecoglossu altivelis), turbot (Scophthalmus maximus) and carp (Cyprinus carpio) (Kanno et al., 1989; Fouz et al., 1990; Lemaitrem et al., 1996). Expression of one or more of the above-mentioned antimicrobial components in fish epidermal mucus has been observed following microbial stress (Aranishi and Mano, 2000; Patrzykat et al., 2001), thus supporting the role of epidermal mucus in protecting fish from infectious pathogens. these preliminary assays indicated that eel fish mucus remain an interesting source for new hemolytic and antibacterial activity with better and also suggest that A. anguilla could be a source of the secondary metabolites with a strong antibacterial activity.


Authors thankful to Prof. Dr. T. Balasubramanian, Director, CAS in Marine Biology, Annamalai University for providing facilities and also thankful to Mr. Muthuraja for collecting fish mucus sample.


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