Identification of Tsetse (Glossina spp.) Using Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry

Antje Hoppenheit,Uwe Roesler,Jayaseelan Murugaiyan,Burkhard Bauer,Stephan Steuber,Peter-Henning Clausen,Rhoel Ramos Dinglasan

doi:10.1371/journal.pntd.0002305

Abstract

Glossina (G.) spp. (Diptera: Glossinidae), known as tsetse flies, are vectors of African trypanosomes that cause sleeping sickness in humans and nagana in domestic livestock. Knowledge on tsetse distribution and accurate species identification help identify potential vector intervention sites. Morphological species identification of tsetse is challenging and sometimes not accurate. The matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) technique, already standardised for microbial identification, could become a standard method for tsetse fly diagnostics. Therefore, a unique spectra reference database was created for five lab-reared species of riverine-, savannah- and forest- type tsetse flies and incorporated with the commercial Biotyper 3.0 database. The standard formic acid/acetonitrile extraction of male and female whole insects and their body parts (head, thorax, abdomen, wings and legs) was used to obtain the flies' proteins. The computed composite correlation index and cluster analysis revealed the suitability of any tsetse body part for a rapid taxonomical identification. Phyloproteomic analysis revealed that the peak patterns of G. brevipalpis differed greatly from the other tsetse. This outcome was comparable to previous theories that they might be considered as a sister group to other tsetse spp. Freshly extracted samples were found to be matched at the species level. However, sex differentiation proved to be less reliable. Similarly processed samples of the common house fly Musca domestica (Diptera: Muscidae; strain: Lei) did not yield any match with the tsetse reference database. The inclusion of additional strains of morphologically defined wild caught flies of known origin and the availability of large-scale mass spectrometry data could facilitate rapid tsetse species identification in the future.

Highlights

The trypanosomiasis infection risk of a particular area is determined by several factors, including tsetse species abundance and the sex distribution of a fly population [1]
Classical species identification relies on minor morphological differences, often challenging for field workers
The ions are accelerated in a vacuum tube, and their Time of Flight (ToF) to reach the detector is recorded

Summary

Introduction

The trypanosomiasis infection risk of a particular area is determined by several factors, including tsetse species abundance and the sex distribution of a fly population [1]. Current tsetse specification based on morphology may not be the only way to rapidly determine the species status of Glossina spp. The matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) is an established method of identification for microorganisms [5,6,7,8,9,10,11]. The MALDIbased identification of microorganisms requires only a small portion of a microbial colony and a drop of matrix solution [12,13,14]. The ions are accelerated into a vacuum tube using a high electric field and the Time of Flight (ToF) to reach the detector is recorded. The technique proved to be time and cost effective, as reliable as genome-based identification methods [6,9].

Objectives

Methods

Results

Discussion

Conclusion