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

International audienceBackground: 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

Anopheles gambiae on remote islands in the Indian Ocean: origins and prospects for malaria elimination by genetic modification of extant populations.

The mosquito Anopheles gambiae s.s. is a primary malaria vector throughout sub-Saharan Africa including the islands of the Comoros archipelago (Anjouan, Grande Comore, Mayotte and Mohéli). These islands are located at the northern end of the Mozambique Channel in eastern Africa. Previous studies have shown a relatively high degree of genetic isolation between the Comoros islands and mainland populations of A. gambiae, but the origin of the island populations remains unclear. Here, we analyzed phylogenetic relationships among island and mainland populations using complete mitochondrial genome sequences of individual A. gambiae specimens. This work augments earlier studies based on analysis of the nuclear genome. We investigated the source population of A. gambiae for each island, estimated the number of introductions, when they occurred and explored evidence for contemporary gene flow between island and mainland populations. These studies are relevant to understanding historical patterns in the dispersal of this important malaria vector and provide information critical to assessing their potential for the exploration of genetic-based vector control methods to eliminate this disease. Phylogenetic analysis and haplotype networks were constructed from mitogenome sequences of 258 A. gambiae from the four islands. In addition, 112 individuals from seven countries across sub-Saharan Africa and Madagascar were included to identify potential source populations. Our results suggest that introduction events of A. gambiae into the Comoros archipelago were rare and recent events and support earlier claims that gene flow between the mainland and these islands is limited. This study is concordant with earlier work suggesting the suitability of these oceanic islands as appropriate sites for conducting field trial releases of genetically engineered mosquitoes (GEMs)

Population Genetics of <i>Anopheles pretoriensis</i> in Grande Comore Island

Anopheles pretoriensis is widely distributed across Africa, including on oceanic islands such as Grande Comore in the Comoros. This species is known to be mostly zoophylic and therefore considered to have low impact on the transmission of human malaria. However, A. pretoriensis has been found infected with Plasmodium, suggesting that it may be epidemiologically important. In the present study, we sequenced and assembled the complete mitogenome of A. pretoriensis and inferred its phylogenetic relationship among other species in the subgenus Cellia. We also investigated the genetic structure of A. pretoriensis populations on Grande Comore Island, and between this island population and sites in continental Africa, using partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Seven haplotypes were found on the island, one of which was ubiquitous. There was no clear divergence between island haplotypes and those found on the continent. The present work contributes knowledge on this understudied, yet abundant, Anopheles species

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

<p>The MS spectra were generated according to two protocols (P1, P2). Intact <i>Ang</i> match the MS profiles from <i>Anopheles gambiae</i> 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 <i>Anopheles gambiae</i> 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.</p

Whatman filter storage method for mosquito blood meal identification.

<p>Comparison of LSVs obtained following a reference TP database query with MS spectra of <i>An</i>. <i>gambiae Giles</i> 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.</p

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

<p>The MS spectrum alignment was performed by Flex analysis 3.3 software. Intact <i>Ang</i> match the MS profiles from <i>Anopheles gambiae</i> 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.</p

Blind tests of the <i>An</i>. <i>gambiae</i> Giles used to set up the protocol optimal for blood meals identification by MALDI TOF from BWFPs.

<p>Blind tests of the <i>An</i>. <i>gambiae</i> Giles used to set up the protocol optimal for blood meals identification by MALDI TOF from BWFPs.</p

Blind tests of Comoros mosquitoes to identified their blood meals sources by MALDI-TOF MS from whatman paper against TPDB.

<p>Blind tests of Comoros mosquitoes to identified their blood meals sources by MALDI-TOF MS from whatman paper against TPDB.</p

<i>An</i>. <i>gambiae</i> Giles abdomen crushing in paper filter Whatman to performed the blind tests according blood meal source.

<p><i>An</i>. <i>gambiae</i> Giles abdomen crushing in paper filter Whatman to performed the blind tests according blood meal source.</p