Impact of Starvation Duration on the Antimicrobial Efficacy and Protein Content of Crude Saliva Extract from Asian Buffalo Leech Hirudinaria Manillensis

Abstract

Hirudinaria manillensis, a freshwater leech endemic to Peninsular Malaysia, exhibits promising antimicrobial and biochemical properties. Its saliva serves as a model for studying annelid biology in leech therapy. This study aimed to assess the protein concentration and antimicrobial activity of crude leech saliva nextracts (CLSE) under varying starvation conditions. CLSE was collected by feeding leeches a phagostimulatory solution through a parafilm membrane. The total protein concentration was measured using the Bradford assay, and antimicrobial activity was evaluated through disc diffusion and minimal inhibitory concentration tests. Our findings indicate that protein production and CLSE secretion are influenced by starvation duration. Protein concentration in CLSE
significantly decreased from 49.8 mg/L ± 0.033 at 2 weeks to 40.34 mg/L ± 0.053 at 4 weeks, with a drastic reduction to 24.45 mg/L ± 0.003 at 8 weeks,
likely due to physiological exhaustion. Microbial activity tests revealed that CLSE collected after a 4 week starvation period exhibited significant inhibition against gram negative strain: Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica, but not Staphylococcus aureus. Beyond the fourth week, no significant changes in inhibitory zones were observed. These results demonstrate that the starvation period significantly affects both protein concentration and antimicrobial efficacy of CLSE. Further investigation into the relationship between starvation duration and protein concentration in relation to microbial activity is necessary. Obtaining sufficient CLSE volumes for comprehensive analysis remains a challenge.