ANTIMICROBIAL ACTIVITY OF MALE AND FEMALE SNAKEHEAD Channa sp. MUCUS AND ITS ASSOCIATED MICROBIOTA AGAINST SELECTED PATHOGENIC BACTERIA OF FRESHWATER FISHES

This study focused on the in vitro antimicrobial activity of the skin mucus (study 1) and the associated mucus microbiota (study 2) of eight snakehead fish against freshwater pathogens as a novel approach for the repertoire of studying the mucosal immune system. Initially for study 1, the total protein content and important immune-related enzymes (i.e., lysozyme and peroxidase) of the crude mucus extracts (CMEs) were measured in respect to the sex of fish. The protein concentration of female CMEs was observed to be significantly higher (p<0.01) than the male CMEs with mean values of 0.41±0.18 mg mL^-1 and 0.37±0.16 mg mL^-1, respectively. Significantly higher (p<0.01) enzymatic activities were also observed on female CMEs compared to male CMEs with mean values of 46.25±14.97 U µL^-1 and 21.75±11.67 U µL^-1, respectively for lysozyme activity, and 1.70±0.85 nmole min^-1 mL and 1.18±0.90 nmole min^-1 mL, respectively for peroxidase activity. Thereafter, the antimicrobial activity of the CMEs was tested through disc-diffusion method against six freshwater fish pathogens viz., Aeromonas hydrophila, Pseudomonas fluorescens, Micrococcus luteus, Enterococcus faecalis, Aspergillus flavus, and Aspergillus niger. The antimicrobial assay against all of the six test pathogens resulted to inhibitions that were primarily exhibited by female CMEs. The sequence for the magnitude of inhibition manifested by female CMEs against the test pathogens was E. faecalis (13.33±1.15 mm) > M. luteus (12.00±0.00 mm) > A. hydrophila (11.33±1.15 mm) > P. fluorescens (10.00±0.00 mm) > A. flavus (10.00±0.00 mm) > A. niger (6.67±0.58 mm). On the other hand, male CMEs exhibited significantly lower (p<0.01) zone of inhibitions compared to female CMEs. The sequence for the magnitude of inhibition manifested by male CMEs against the test pathogens was E. faecalis (12.33±1.53 mm) > A. hydrophila (10.00±0.00 mm) > A. flavus (10.00±0.00 mm) > M. luteus (9.67±0.58 mm).

For study 2, the resident mucus bacteria were isolated from fresh mucus and were purified thrice. The mucus bacterial isolates (MBIs) were classified through Gram classification and protease production and was further identified through 16S rRNA gene sequencing. The 16 MBIs were classified and identified to be under the genus Bacillus, Acinetobacter, Lysinibacillus, Aeromonas and Staphylococcus. According to their phylogeny, some MBIs were found to be closely related species. On the other hand, the antimicrobial activity of the MBIs against the six test pathogens resulted to positive inhibitions as exhibited by three Bacillus spp. isolates (i.e., M1_1, M2_1, and M2_2).

The protein concentrations and enzymatic activities were positively correlated with the antimicrobial activities of CMEs. Moreover, the more antagonistic female CMEs may be a potential source of antimicrobial agents. Also, some MBIs exhibited antimicrobial activities suggesting their possible antagonistic roles in the mucus. Thus, further studies for the utilization of the mucus and its microbiota may lead to novel immunotherapies.