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Background  Black flies (Diptera: Simuliidae) are haematophagous insects that can cause allergic reactions and act as vectors of pathogens. Although their saliva has been thought to contain a diverse array of physiologically active molecules, little information is available on antimicrobial factors in black fly salivary glands, especially no defensins have been reported so far.    Methods  A novel cationic defensin designated  Siba Def was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly  Simulium bannaense . The amino acid sequence of  Siba Def was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-positive bacteria  Staphylococcus aureus  or  Bacillus subtilis  treated with  Siba Def were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the expression of  Siba Def mRNA in whole bodies of insects after oral infection with the bacteria  S. aureus  or  B. subtilis .    Results  Surprisingly, the phylogenetic analysis of defensin-related amino acid sequences demonstrated that  Siba Def is most closely related to defensins from the human body louse  Pediculus humanus corporis  (Anoplura: Pediculidae), rather than to other dipteran defensins.  Siba Def showed potent antimicrobial activities against Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.83 μM to 2.29 μM. SEM analysis indicated that  Siba Def killed microorganisms through the disruption of cell membrane integrity. The transcript levels of  Siba Def in the bacteria-immunized flies increased with the time course, reaching maximum at 36 h and then slowly decreased from that time point.    Conclusions  Our results indicate that  Siba Def is involved in the innate humoral response of the black fly  S. bannaense , and it might play a significant role in the defence against microorganisms in both sugar and blood meals.

Purification and characterization of a novel defensin from the salivary glands of the black fly, Simulium bannaense