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

Lin Wei,Yipeng Wang,Lixian Mu,Hailong Yang,Jing Lv,Cuiping Feng,Jun Li,Jing Wu,Hui Bian,Yiling Lu,Tong Liu,Yi Han

doi:10.1186/s13071-015-0669-9

Abstract

BackgroundBlack 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.MethodsA novel cationic defensin designated SibaDef 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 SibaDef 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 SibaDef were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the expression of SibaDef mRNA in whole bodies of insects after oral infection with the bacteria S. aureus or B. subtilis.ResultsSurprisingly, the phylogenetic analysis of defensin-related amino acid sequences demonstrated that SibaDef is most closely related to defensins from the human body louse Pediculus humanus corporis (Anoplura: Pediculidae), rather than to other dipteran defensins. SibaDef 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 SibaDef killed microorganisms through the disruption of cell membrane integrity. The transcript levels of SibaDef in the bacteria-immunized flies increased with the time course, reaching maximum at 36 h and then slowly decreased from that time point.ConclusionsOur results indicate that SibaDef 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.

Highlights

Black flies (Diptera: Simuliidae) are haematophagous insects that can cause allergic reactions and act as vectors of pathogens
No antimicrobial peptide (AMP) belonging to the defensin family has so far been biochemically characterized from black fly
The cDNA encoding protein precursor is composed of 105 amino acid residues, including a predicted 22 amino acid signal peptide, a 37 amino acid propeptide region and a 46 amino acid mature SibaDef peptide

Summary

Introduction

Black flies (Diptera: Simuliidae) are haematophagous insects that can cause allergic reactions and act as vectors of pathogens 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. As an important hematophagous arthropod, there was not much information available about pharmacologically active compounds in black fly salivary glands, until salivary transcriptomes have been made and described from three black fly species (S. guianense, S. vittatum and S. nigrimanum) [19,20,21] In these studies, many more substances with potential anti-haemostatic functions or immunity-related activities have been uncovered. No antimicrobial peptide (AMP) belonging to the defensin family has so far been biochemically characterized from black fly

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