MALDI-TOF MS: An effective tool for a global surveillance of dengue vector species

Abstract

Dengue, Zika and chikungunya viruses cause significant human public health burdens in the world. These arboviruses are transmitted by vector mosquito species notably Aedes aegypti and Aedes albopictus. In the Pacific region, more vector species of arboviruses belonging to the Scutellaris Group are present. Due to the expansion of human travel and international trade, the threat of their dispersal in other world regions is on the rise. Strengthening of entomological surveillance ensuring rapid detection of introduced vector species is therefore required in order to avoid their establishment and the risk of arbovirus outbreaks. This surveillance relies on accurate species identification. The aim of this study was to assess the use of the Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) as a tool for an international identification and surveillance of these mosquito vectors of arboviruses. Field-mosquitoes belonging to 8 species (Ae. aegypti, Ae. albopictus, Aedes polynesiensis, Aedes scutellaris, Aedes pseudoscutellaris, Aedes malayensis, Aedes futunae and Culex quinquefasciatus) from 6 countries in the Pacific, Asian and Madagascar, were included in this study. Analysis provided evidence that a MALDI-TOF database created using mosquitoes from the Pacific region allowed suitable identification of mosquito species from the other regions. This technic was as efficient as the DNA sequencing method in identifying mosquito species. Indeed, with the exception of two Ae. pseudoscutellaris, an exact species identification was obtained for all individual mosquitoes. These findings highlight that the MALDI-TOF MS is a promising tool that could be used for a global comprehensive arbovirus vector surveillance.

Fig 1. Comparison of the results obtained with morphological identification and MALDI‑TOF analysis for the Aedes species.

X‑axis corresponds to the results obtained with MALDI‑TOF analysis. Y‑axis corresponds to Log-score values (LSVs). Colors illustrate morphological identification obtained through double‑blind evaluation and confirmed with DNA sequencing for the Aedes of the Scutellaris Group. Black dashed-line corresponds to the threshold value for mosquito species identification, stated during previous study [29]. Red dashed-line corresponds to the threshold value for the identification of mosquito species belonging to the Scutellaris Group.

Fig 2. Identification scores of mosquitoes collected from the three regions when using the Pacific region MALDI-TOF database.

Three mosquito species were analyzed: Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. Colors illustrate the region of collection. Each boxplot consists of nine to 40 LSVs per species and per region. Black dashed-line corresponds to the threshold value for mosquito species identification, stated during previous study [29]. Red dashed-line corresponds to the threshold value for the identification of mosquito species belonging to the Scutellaris Group. Wilcoxon test, *p-value < 0.05, ***p-value < 0.001. Abbreviation: NS, not significant.

Fig 3. Maximum likelihood evolutionary (MLE) relationships of mosquito species of the Scutellaris Group.

Tree derived from 45 concatenated sequences. For analysis, the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown for values over 80. The country of collection is indicated along with the specimen number. Colors indicate mosquito species.

Fig 4. Maximum likelihood evolutionary (MLE) relationships of mosquito species of the Scutellaris Group.

A—Maximum likelihood tree derived from 49 cox1 nucleotide sequences. Forty sequences generated during the current study (in black), six sequences generated during our previous study and deposited in the GenBank database (in black) [29] and three sequences retrieved from the GenBank database (in blue). B—Maximum likelihood tree derived from 49 ITS2 nucleotide sequences. Forty-five sequences were generated during this study (in black) and four sequences were retrieved from the GenBank database (in blue). For all analyses, the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown for values over 80. The country of collection is indicated along with the specimen number. Colors indicate mosquito species.

Fig 5. MSP dendrogram of all the mosquitoes belonging to the Scutellaris Group in the MALDI‑TOF database.

The two Aedes aegypti was used as outgroup taxa. Colors of the branches indicate mosquito species according to the cut-off of 500 (arbitrary units) previously described [41].