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

<div><p><i>Glossina (G.)</i> 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 <i>G. brevipalpis</i> 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 <i>Musca dome</i>stica (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.</p></div

Laboratory-reared <i>Glossina (G.)</i> spp. selected for the compilation of spectra database.

1<p>Tsetse & Trypanosomiasis Research Institute, Tanga, Tanzania;</p>2<p>International Atomic Energy Agency, Seibersdorf, Austria.</p

Score-oriented main spectra dendrogram of whole <i>Glossina spp</i>. extracts.

<p>The dendrogram was calculated by Biotyper 3.0 software with distance measure set at correlation and linkage set at complete.</p

Spectra reproducibility among the biological and technical replicates.

<p>Overlay view of 27 spectra obtained from biological and technical replicates of <i>Glossina austeni</i> female whole insect. The masses (in Da) of the ions are shown on the <i>x</i>-axis and the <i>m/z</i> value stands for mass to charge ratio. On the y-axis, the relative intensity of the ions (a.u., arbitrary units) is shown. In the insert, zoomed m/z 5000 to 5200 displays the uniformity among the measured spectra and the stacked view m/z 9000 to 12500 provides a direct comparison of all 27 measured spectra.</p

Representative spectra from the whole insect and different body parts of female <i>Glossina austeni</i>.

<p>Peak pattern of whole and body parts extractions of <i>Glossina austeni</i> female. The x-axis <i>m/z</i> values represent the mass to charge ratio and on the y-axis the relative intensity of the ions (a.u., arbitrary units) is shown. A) Whole insect, B) abdomen, C) head, D) legs, E) thorax and F) wings.</p

Representative spectra of whole insect extraction of male and female <i>Glossina</i> spp.

<p>Mass spectra peak pattern of whole insect extractions of male and female <i>Glossina (G.)</i> spp. The x-axis <i>m/z</i> value stands for mass to charge ratio and the relative intensity of the ions (a.u., arbitrary units) is shown on the y-axis. A) <i>G. morsitans morsitans</i> female, B) <i>G. morsitans morsitans</i> male, C) <i>G. austeni</i> female, D) <i>G. austeni</i> male, E) <i>G. pallidipes</i> female F) <i>G. pallidipes</i> male, G) <i>G. palpalis gambiensis</i> female, H) <i>G. palpalis gambiensis</i> male, I) <i>G. brevipalpis</i> female, and J) <i>G. brevipalpis</i> male.</p

Composite correlation index of tsetse spectra sets.

<p>Evaluation of uniqueness among the spectra sets of 60 tsetse spectra measurements of male (M) and female (F) individuals and their body parts. Composite correlation index matrix was calculated with Biotyper 3.0 software in the mass range of 3000–12000 Da, resolution 4, 4 intervals and auto-correction off. Red indicates relatedness between the spectra sets and dark green indicates incongruence.</p