Identification of French Guiana anopheline mosquitoes by MALDI-TOF MS profiling using protein signatures from two body parts

Abstract

In French Guiana, the malaria, a parasitic infection transmitted by Anopheline mosquitoes, remains a disease of public health importance. To prevent malaria transmission, the main effective way remains Anopheles control. For an effective control, accurate Anopheles species identification is indispensable to distinguish malaria vectors from non-vectors. Although, morphological and molecular methods are largely used, an innovative tool, based on protein pattern comparisons, the Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) profiling, emerged this last decade for arthropod identification. However, the limited mosquito fauna diversity of reference MS spectra remains one of the main drawback for its large usage. The aim of the present study was then to create and to share reference MS spectra for the identification of French Guiana Anopheline species. A total of eight distinct Anopheles species, among which four are malaria vectors, were collected in 6 areas. To improve Anopheles identification, two body parts, legs and thoraxes, were independently submitted to MS for the creation of respective reference MS spectra database (DB). This study underlined that double checking by MS enhanced the Anopheles identification confidence and rate of reliable classification. The sharing of this reference MS spectra DB should make easier Anopheles species monitoring in endemic malaria area to help malaria vector control or elimination programs.

Enhanced procedures for mosquito identification by MALDI-TOF MS

“…To improve mosquito identi cation, notably for sibling or cryptic species, it was proposed to submit to MS, independently, two distinct body parts from the same specimen. This strategy applied to the thoraxes and legs of mosquitoes allowed to corroborate the identi cation obtained per body part and enhanced the identi cation con dence level [32,33]. The same strategy applied to ticks permitted the classi cation of unambiguously closely related Ixodes species [21].…”

“…Although mosquito legs were the body part the most frequently used for specimen identi cation by MS analysis [26, 27,30,31], for which a standardized protocol was established [29][44], other anatomic parts were selected, including thoraxes [32,33], cephalothoraxes [34,35] or heads [24]. These last body parts were chosen, notably, to prevent the risk of non-identi cation from specimens having lost all their breakable legs.…”

“…The calculation of this score, which ranged from 0 to 3, was done using a biostatistical algorithm from the MALDI Biotyper software v.3.0. According to previous studies [33,43,44], LSVs greater than 1.8 were considered reliable for species identi cation. Data were analyzed by R software (R core Team, R Foundation for Statistical Computing, Vienna, Austria).…”

“…The quantity of mix buffer added for homogenization was body part dependent. Respectively, 30µL and 50 µL of mix buffer were used for legs and thoraxes, as previously described [32,33], and 30µL of mix buffer were used for heads based on our preliminary tests. For themanual homogenization, the samples were ground either with MP (with tips of 10µl) or PP until completed homogenization as previously described [24,41].…”

“…To circumvent this limitation, the thoraxes were recently proposed as a complementary body part for mosquito identi cation by MS pro ling [32]. The submission of these two body parts to MS pro ling demonstrated a corroboration of species identi cation, reinforcing identi cation con dence and success rate [32,33].…”