MALDI-TOF MS identification of Anopheles gambiae Giles blood meal crushed on Whatman filter papers

Abstract

Background: Identification of the source of mosquito blood meals is an important component for disease control and surveillance. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as an effective tool for mosquito blood meal identification, using the abdomens of freshly engorged mosquitoes. In the field, mosquito abdomens are crushed on Whatman filter papers to determine the host feeding patterns by identifying the origin of their blood meals. The aim of this study was to test whether crushing engorged mosquito abdomens on Whatman filter papers was compatible with MALDI-TOF MS for mosquito blood meal identification. Both laboratory reared and field collected mosquitoes were tested.

Material and methods: Sixty Anopheles gambiae Giles were experimentally engorged on the blood of six distinct vertebrate hosts (human, sheep, rabbit, dog, chicken and rat). The engorged mosquito abdomens were crushed on Whatman filter papers for MALDI-TOF MS analysis. 150 Whatman filter papers, with mosquitoes engorged on cow and goat blood, were preserved. A total of 77 engorged mosquito abdomens collected in the Comoros Islands and crushed on Whatman filter papers were tested with MALDI-TOF MS.

Results: The MS profiles generated from mosquito engorged abdomens crushed on Whatman filter papers exhibited high reproducibility according to the original host blood. The blood meal host was correctly identified from mosquito abdomens crushed on Whatman filter papers by MALDI-TOF MS. The MS spectra obtained after storage were stable regardless of the room temperature and whether or not they were frozen. The MS profiles were reproducible for up to three months. For the Comoros samples, 70/77 quality MS spectra were obtained and matched with human blood spectra. This was confirmed by molecular tools.

Conclusion: The results demonstrated that MALDI-TOF MS could identify mosquito blood meals from Whatman filter papers collected in the field during entomological surveys. The application of MALDI-TOF MS has proved to be rapid and successful, making it a new and efficient tool for mosquito-borne disease surveillance.

Fig 1. MALDI-TOF MS spectra from An. gambiae Giles (Ang) abdomen protein extracts engorged on vertebrate host bloods and then crushed on Whatman filter papers (WFPs).

The MS spectra were generated according to two protocols (P1, P2). Intact Ang match the MS profiles from Anopheles gambiae Giles abdomens crushed on WFPs. The blood-free WFP corresponds to the MS profiles of WFPs where no mosquito blood meal was released. The vertebrate host bloods used for Anopheles gambiae Giles bloody Whatman filter papers (bloody WFPs) were human and sheep. All mosquitoes were collected 12 hours after feeding. a.u. arbitrary units; m/z mass-to-charge ratio.

Fig 2. Comparison of MALDI-TOF MS spectra from An. gambiae Giles abdomen protein extracts engorged on vertebrate host bloods and then crushed on Whatman filters.

The MS spectrum alignment was performed by Flex analysis 3.3 software. Intact Ang match the MS profiles from Anopheles gambiae Giles abdomens crushed on WFPs. The vertebrate host bloods used for mosquito blood meals were rabbit, dog, chicken and rat. All mosquitoes were collected 12 hours after feeding. a.u. arbitrary units; m/z mass-to-charge ratio.

Fig 3. Whatman filter storage method for mosquito blood meal identification.

Comparison of LSVs obtained following a reference TP database query with MS spectra of An. gambiae Giles fed on cow and goat blood. All specimens were collected 12 hours after feeding and stored up to 90 days (D). The mosquito abdominal proteins crushed on WFPs were stored under three different conditions: -20°C, + 4°C and room temperature. The dashed line represents the threshold value for relevant identification (LSVs >1.8). LSV, log score value.